Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary System

Toxicologic Pathology, 38: 5S-81S, 2010 Copyright # 2010 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623310386499

1 2* 3 4 5 6 BOB THOOLEN ,ROBERT R. MARONPOT ,TAKANORI HARADA ,ABRAHAM NYSKA ,COLIN ROUSSEAUX ,THOMAS NOLTE , 7 8 9 10 11 12 DAVID E. MALARKEY ,WOLFGANG KAUFMANN ,KARIN KU¨ TTLER ,ULRICH DESCHL ,DAI NAKAE ,RICHARD GREGSON , 13 14 15 16 17 MICHAEL P. VINLOVE ,AMY E. BRIX ,BHANU SINGH ,FIORELLA BELPOGGI , AND JERROLD M. WARD 1Global Pathology Support, The Hague, The Netherlands 2Maronpot Consulting LLC, Raleigh, North Carolina, USA 3The Institute of Environmental Toxicology, Joso-shi, Ibaraki, Japan 4Haharuv 18, Timrat, Israel 5Wakefield QC, Canada 6Boehringer Ingelheim Pharma GmbH & Co., Biberach an der Riss, Germany 7National Toxicology Program, Cellular and Molecular Pathology Branch, Research Triangle Park, North Carolina, USA 8Merck KGaA, Darmstadt, Germany 9BASF Aktiengesellschaft, Ludwigshafen, Germany 10Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach/Riss, Germany 11Tokyo Metropolitan Institute of Public Health, Shinjuku, Tokyo, Japan 12Charles River Laboratories, Pathology Department, Senneville, QC, Canada 13Pathology Associates, Charles River, Frederick, Maryland, USA 14Experimental Pathology Laboratories Inc., Research Triangle Park, North Carolina, USA 15DuPont Haskell Global Centers for Health and Environmental Science, Newark, Delaware, USA 16Ramazzini Institute, Bentivoglio (BO), Italy 17Global VetPathology, Montgomery Village, Maryland, USA *Chairman of the Liver INHAND Committee

ABSTRACT

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an interna- tionally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and differential diagnosis for classifying microscopic lesions observed in the hepatobiliary system of laboratory rats and mice, with color microphotographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available for society members electronically on the internet (http://goreni.org). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions of the hepatobiliary system in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Keywords: diagnostic pathology; hepatobiliary system; histopathology; liver; nomenclature; rodent pathology.

Address correspondence to: Bob Thoolen, Global Pathology Support, Benoordenhoutseweg 23, The Hague 2596 BA, Netherlands; e-mail: bob.thoolen@ gpstoxpath.com. Financial Disclosure: No money was paid for the preparation of this manuscript. During the construction of this manuscript salaries of contributors were paid by their respective companies. None of the content of the manuscript contains any information that could be patentable or claimed as intellectual property of the contributors or their respective companies. Abbreviations: AE1/AE3, Two clones of anti-cytokeratin monoclonal antibodies; a.k.a., Also known as; AS, Anterior Segment; a-SMA, a-smooth muscle actin; Bcl-2, B-cel lymphoma 2 - apoptosis regulator protein; BSTP, British Society of Toxicological Pathologists; CD (31, 34, 68), Cluster differentiation (31, 34, 68); CEA, Carcinoembryonic antigen; CK, Cytokeratin; ED1, Rat homologue of human CD68; EM, Electron microscopy; ESTP, European Society of Toxicologic Pathology; Factor VIII, Blood clotting factor/anti-hemophilic factor; F4/80, Rat anti-mouse macrophage monoclonal antibody; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; JSTP, The Japanese Society of Toxicologic Pathology; Ki-67, Nuclear protein associated with proliferation; LAMP, Lysosome- associated protein; LLL, Lef lateral lobe; LML, Left medial lobe; MIB-1, Monoclonal antibody that detects K-67 antigen on formalin fixed paraffin embedded sections; MS, Middle Segment; NTP, National Toxicology Program; NLDC-145, Rat anti-mouse dendritic cell monoclonal antibody; NOS, Not otherwise specified; OX-6, MHC Class II Ia antibody; PAS, Periodic acid-Schiff; PC, Caudate Process; PCNA, Proliferator Cell Nuclear Antigen; PCR, Polymerase Chain Reaction; PP, Papillary Process; PPA, Processus papillaris anterior; PPAR, Peroxisome Proliferator-Activated Receptor; PS, Posterior Segment; RER, Rough Endoplasmic Reticulum; RLL, Right lateral lobe; RML, Right medial lobe; SRA-E5, Mouse monoclonal anti-macrophage antibody for Scavenger Receptor A; SOPs, Standard Operating Procedures; STP, Society of Toxicologic Pathology.

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TABLE 1.—Species differences in liver lobes.

Human (4 / 8)a Monkey (4 / 8) Dog (6 / 7) Rat (4 / 7) Mouse (4 / 7) Cat (6 / 7) Left liver Left Lobe (2 segments) Left (lateral) Lobe Left Lobe Left Lobe Left Lobe Left Lobe LLL AS þ PS LLL þ LML LLL þ LML LLL þ LML (largest) þ LML Right liver Right Lobe (2 segments) Right (lateral) Lobe Right Lobe RLL Right Lobe Right Lobe Right Lobe AS þ MS þ PS (impression) þ RML RLL þ RML RLL þ RML RLL þ RML Intermediate liver Quadrate Lobe Median Lobe (largest) Quadrate Lobe Quadrate Lobe Quadrate Lobe (small) Caudate lobe Caudate Lobe Caudate Lobe Caudate Lobe Caudate Lobe Caudate Lobe Caudate Lobe PC þ PP PP þ PC PP þ PPA þ PC PP þ PC PP þ PC

LLL ¼ Left lateral lobe; RLL ¼ Right lateral lobe; PC ¼ Caudate Process; LML ¼ Left medial lobe; RML ¼ Right medial lobe; PP ¼ Papillary Process; PPA ¼ Processus papillaris anterior; AS ¼ Anterior Segment; MS ¼ Middle Segment; PS ¼ Posterior Segment. Gray, Williams, and Bannister (1995); Browning, Schroeder, and Berringer (1974); Ko¨nig, Sautet, and Liebich (2004); Rajtova´, Hora´k, and Popesko (2002); Vons et al., 2009. a (Number of lobes / Number of lobes including segments).

I. GENERAL INTRODUCTION frequently observed in pathological evaluation of toxicity studies. The liver is a major target organ in safety assessment of preclinical toxicity and oncogenicity studies with rodents; hence, hepatic pathology is central to many toxicological II. ANATOMY pathology studies. As toxicologic pathologists sometimes The liver occupies the cranial third of the abdominal experience difficulties in distinguishing the wide variety of cavity and is comprised of multiple lobes; however, the liver lesions in the rodents for safety evaluation purposes, this nomenclature for the liver lobes varies among authors. There document is a consensus of senior toxicologic pathologists are basically left, middle, right,andcaudate lobes (Harada regarding suggested nomenclature that should be used for et al. 1999; Eustis et al. 1990). A thin connective tissue cap- specific lesions. sule that is externally lined by peritoneal mesothelial cells Standardized diagnostic criteria and nomenclature are covers the parietal and visceral surfaces of the liver. The essential to harmonize the classification and reporting of hepa- middle lobe has an incomplete fissure where the falciform tic nonproliferative as well as proliferative lesions. This ligament attaches. In mice the gallbladder is located in the INHAND document serves as a framework that can be used for middle lobe fissure, whereas the rat does not have a gallblad- the harmonization of diagnostic criteria of hepatic lesions in der. The right lobe has an anterior and posterior component laboratory rats and mice. These recommendations for diagnos- and the small caudate lobe consists of two or more disc- tic criteria and preferred terminology should not be considered like sublobes (See Figure 1). mandatory; proper diagnoses are ultimately based on the dis- Nomenclature for liver lobes varies among species and cretion of the toxicologic study pathologist. sometimes among authors. A table showing differences in liver The INHAND (International Harmonization of Nomenclature lobes between species is included based on current anatomic and Diagnostic Criteria for Lesions in Rats and Mice) initiative features (Table 1). creates a framework for the harmonization of diagnostic nomenclature (classification of lesions using the same terminology) in III. HISTOMORPHOLOGY different rodent organ systems. It is a joint initiative between Societies from the United States (STP), Great Britain (BSTP), The two-dimensional microarchitecture of the liver has Japan (JSTP), and European countries (ESTP). been categorized in at least three perspectives (Figure 2). The This document is organized to provide introductory material anatomic model is the classical lobule, a hexagonal structure that reviews comparative interspecies differences in anatomy divided into concentric centrilobular, midzonal, and peripor- and liver function, followed by a listing of liver lesions in a tal segments. The triangular portal lobule is based on bile standardized format. The liver lesions descriptions include dif- flow and is centered on the portal triad (portal canal). The ferential diagnoses to aid in distinguishing primary diagnoses elliptical or diamond shaped liver acinus is a functional sub- from similar appearing lesions. Throughout the document, unit of the liver. It incorporates blood flow and metabolic comparisons are made with respect to similar liver lesions that functions and is divided in zone 1 (periportal), zone 2 (transi- may occur in humans. It should be noted that the preferred diag- tional; midzonal), and zone 3 (centrilobular). Functionally, nostic terminology for some lesions in this document might zone 1 hepatocytes are specialized for oxidative liver func- represent departures from traditional nomenclature schemes tions such as gluconeogenesis, b-oxidation of fatty acids, and found in standard textbooks. Furthermore, illustrative photomi- cholesterol synthesis, while zone 3 cells are more important crographs for a given diagnostic entity may occasionally depict for glycolysis, lipogenesis, and cytochrome P-450–based additional tissue changes as this reflects actual situations drug detoxification.

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1. Blood Supply and Bile Flow lymphocytes that have natural killer activity and are primarily located in periportal areas (Wright and Stacey 1991). The liver has a dual blood supply, the hepatic portal vein and the hepatic artery. The hepatic artery supplies oxygenated 3. Immunohistochemistry blood. Approximately 75% of the blood is delivered to the liver via the hepatic portal vein that drains the spleen, stomach, Immunohistochemistry (IHC), utilizing fluorescent or chro- intestines, and pancreas. Branches of the hepatic artery and mogen tagged antibodies, is a useful adjunct for identification portal vein are seen in the portal triads along with bile ducts and of different cell types in the liver. Selected examples are pro- are separated from the hepatic cords by a ‘‘limiting plate’’ of vided in Table 2. hepatocytes. The bile ducts join to form the hepatic duct lead- Use of IHC can be helpful for diagnostic purposes and is ing to the small intestine in rats and to the gallbladder in mice. common in human pathology where panels of immunohisto- Blood flows from the portal areas to the central vein in the cen- chemical stains are used for supporting diagnoses. Not all com- ter of each lobule while bile flows from the center of the hepa- mercially available preparations of a given antibody will react tic lobule to the portal areas and on to the hepatic duct. the same way between different laboratories and between different species. Furthermore, expertise is required for 2. Histology tissue handling to unmask cellular antigens that may be cross-linked during tissue fixation. Diagnostic evaluation of The two most commonly used descriptions for the structural immunostains typically requires inclusion of both positive and and functional units of the liver are the hepatic lobule (Kiernan negative controls. The interpretations of IHC results are usually 1883) and the acinus (Rappaport et al., 1954) (Figure 2). The performed in conjunction with histopathological findings and structural unit, the hepatic lobule, is modeled on the blood flow sometimes also with consideration of gross findings and/or within the liver and is commonly used for descriptive pathol- clinical pathology or other relevant study results. ogy and morphological diagnoses. The functional unit, the hepatic acinus, is modeled on blood flow and metabolism IV. PHYSIOLOGY within the liver. More recently a parenchymal unit in the liver has been described as a cone-shaped three-dimensional struc- The liver is responsible for maintenance of many homeo- ture comprised of approximately fourteen hepatic lobules sup- static and physiological functions. Liver size is governed both plied and drained by common vascular tributaries (Malarkey by genetic factors and by the rate of biochemical activity to et al. 2005; Teutsch, Schuerfeld, and Groezinger 1999; Teutsch maintain optimal functional mass. It is an organ system capable 2005). This parenchymal unit more closely explains the ran- of rapid responses to a variety of noxious stimuli. Following dom size and shape distribution of the more classical hepatic loss of hepatocytes from stimuli such as transient toxic insult, lobule as seen in a conventional two-dimensional histology infection, or partial hepatectomy, the liver is rapidly restored slide. It also provides a basis for understanding the heteroge- to its optimal mass to maintain normal function. neous response of various hepatic lobules to chemical insult. Liver functions are complex and diverse including endo- In addition to hepatocytes, the liver is comprised of a variety crine and exocrine activity, metabolism, conjugation, detoxifi- of cell types, including biliary cells, endothelial cells, Kupffer cation, and hematopoiesis in early embryonic and fetal cells, Ito cells (stellate cells), fat-storing cells, and pit cells in development (Harada et al. 1999). The liver is continuously addition to hematopoietic cells in the sinusoids and blood ves- exposed to all ingested substances absorbed through the intest- sels. Polyhedral hepatocytes comprise approximately 60% of inal tract via the portal vein and systemically via the arterial the liver arranged in plates or cords that radiate from the central blood supply. A pivotal hepatic function in toxicologic pathol- vein to the portal areas. In two-dimensional sections they are ogy is xenobiotic biotransformation that leads to detoxification typically one cell layer thick and form anastomoses (Miyai of materials absorbed in the intestinal tract. Xenobiotic 1991). On one surface they are separated from the sinusoidal metabolism by hepatocytes can occur by phase I (often the wall by a peri-sinusoidal space, the space of Disse, where cytochrome oxidase series) and phase II reactions (often the they are exposed to tissue fluids. On the opposite side of formation of the water soluble glucuronide) (Graham and Lake the hepatocyte bile canaliculi are formed with hepatocytes in 2008; Martignoni, Groothuis, and de Kanter 2006). Hepatic an adjacent hepatic cord. Desmosomes, gap junctions, and metabolic processes may also cause indirect toxicity by gener- stud-like protrusions connect contiguous hepatocytes within a ating electrophilic species capable of reacting with proteins, cord. Biliary cells form bile ducts in the portal areas and nucleic acids, and other cytoplasmic organelles (Xu, Li, and constitute the portal triad with a hepatic artery and a portal Kong 2005). Intrinsic and induced enzymes responsible for vein. Fenestrated endothelial cells line the sinusoids and hepatic function may be unevenly distributed throughout the synthesize prostaglandins. Kupffer cells are a self-renewing hepatic lobule and between the different lobes (Greaves 2007). fixed macrophage comprising approximately 10% of all liver The presence of background changes and undercurrent dis- cells (Eustis et al. 1990). Kupffer cells are phagocytic, secrete ease states affects the hepatic and biliary morphology, for mediators of inflammation, and catabolize lipids and proteins. Ito example, caloric restriction diminishes hepatocellular size and cells (stellate cells) are peri-sinusoidal cells that store vitamin A can make interpretation of test-article–related changes more and are also a major source of collagen in the liver. Pit cells are challenging. Other factors that influence the liver morphology

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TABLE 2.—Selected immunohistochemical stains that have been Most toxicologic pathologists use a common grading scale used to identify different cell types in liver sections. such as marginal or minimal, slight, moderate, marked, and severe for inflammatory, necrotizing, or other degenerative and Immunohistochemical stains of liver cells responsive lesions. Tissue-specific locators are often used, such Cell type Antibody as portal, periportal, midzonal, centrilobular, hilar, ductal, peri- ductal, peri-canalicular, or subcapsular to indicate the lesion Hepatocytes CK8, CK18 distribution within the liver. Focal, multifocal, and diffuse are Bile canaliculi Polyclonal CEA Bile duct epithelium CK7, CK19, AE1/AE3 commonly used modifiers in the morphological diagnosis for Endothelial cell Factor VIII, CD31, CD34 distribution parameters. Based on the formal definition, a focal Exudate macrophages (monocytes) ED1 lesion refers to one specific area, or focus, whereas multifocal Kupffer cells CD68, F4/80, ED2, SRA-E5 refers to more than one focus (foci). However, some patholo- Hepatic stellate cells (activated), a-SMA gists use focal for both focal and multifocal, referring to the myofibroblasts and smooth muscle cells Dendritic cells NLDC-145, OX-6 nature of the lesion rather than its actual distribution and using Oval cells a-fetoprotein (AFP), CK20 grading to reflect the extent of the multifocality. Schemes for Apoptosis Bcl-2, Caspase 3 and 7 scoring lesion severity vary widely and no single system is Proliferation markers Ki67/MIB-1, PCNA likely to be accepted by all pathologists. While a sample grading scheme for focal and multifocal liver lesions is provided in Geller, Dahll, and Alsabeh (2008); Malhotra, Sakhuja, and Gondal (2004); Hurlimann and Gardiol (1991); Davenport et al. (2001); Kashiwagi, Kaidoh, and Inoue´(2001); Faa Table 3, this should not be regarded as a universal or specific et al. (1998). INHAND-recommended grading scheme. are: body weight loss, blood flow, food intake, vascular and TABLE 3.—A sample grading scheme for focal and multifocal hemodynamic changes, timing and duration of exposure, with- liver lesions (modified from Hardisty and Eustis 1990; World drawal effects, and functional heterogeneity. Functional hetero- Health Organization 1978; Derelanko 2000). geneity expresses itself via differences in metabolism, oxygen supply, b-oxidation, amino acid metabolism, gluconeogenesis, Proportion of Quantifiable glycolysis, ureagenesis, liponeogenesis, and bile acid and biliru- Severity liver affected Grade finding bin secretion. These factors can affect occurrence of nonproli- Marginal or minimal Very small amount 1 1-2 foci ferative as well as proliferative liver lesions in rodents. Slight or few Small amount 2 3-6 foci Moderate or several Medium amount 3 7-12 foci Marked or many Large amount 4 >12 foci V. LIVER NECROPSY AND TRIMMING PROTOCOL Severe Very large amount 5 Diffuse At necropsy, rat and mouse liver may be weighed and individual liver lobes examined carefully for gross lesions. In VII. NOMENCLATURE,DIAGNOSTIC CRITERIA, AND DIFFERENTIAL conventional preclinical rodent studies, gross lesions must be DIAGNOSIS correlated with the histopathological findings. Liver-specific A. Congenital Lesions trimming protocols (see Figure 1) according to standard operating procedures (SOPs) are used (e.g., see Ruehl-Fehlert Introduction et al. 2003). Dissected lobes and trimmed liver pieces can be Developmental anomalies occasionally occur in the liver of fixed in 10% neutral buffered formalin (no more than 1 cm rodents. These malformations might be expressed in different thick in 1:10 tissue: formalin). forms and be of different origin. They mostly occur as isolated effects and are considered by the pathologist in distinguishing VI. GRADING OF LIVER LESIONS background hepatic lesions versus xenobiotic-induced lesions Interpretation of hepatic lesions in safety assessment studies that occur in rodent preclinical toxicity studies. requires consideration of gross and microscopic findings, hematology, clinical chemistry, and liver weights in the con- Hepatodiaphragmatic Nodule (Figures 3 and 4) current control groups of animals and should take into account Pathogenesis: Developmental alteration. species and strain, age, caging, diet, and tissue sampling. Many pathologists use a grading system to document lesion Diagnostic features: severity. In toxicological pathology, the generation of ordinal data using a scoring system allows statistical analysis for Visible grossly and tinctorially similar to normal effects and trends (Gad and Rousseaux 2002). However, not all hepatic parenchyma. grading systems are the same and may differ in how they incor- Rounded extensions usually of the medial lobe(s). porate distribution, stage, and extent of lesions. The problem of Increased mitoses, cytological alterations, and harmonization as it relates to lesion severity has been recog- nuclear alterations may be present. nized and discussed in some detail (Hardisty and Eustis Linear chromatin structures with small lateral projec- 1990; World Health Organization 1978). tions are pathognostic.

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Differential diagnosis: adaptive changes usually do not result in illness or death of rodents. Often these processes are dose and chemical related. Hepatocellular focus of cellular alteration—tinctorial variation from normal parenchyma and does not protrude into the diaphragm. Fatty Change Hepatocellular neoplasia—when visible grossly does Synonyms/subtypes: Lipidosis, vacuolation, lipid, macrovesi- not protrude into the thoracic cavity. cular and/or microvesicular steatosis, phospholipidosis.1 Regenerative hyperplastic nodule (nodular hyperplasia)—typically involves multiple nodules of Pathogenesis: Perturbations in lipid metabolism and disposition. hyperplasia separated by proliferative bands of oval cells or connective tissue. Diagnostic features: Macrovesicular fatty change (Figures 5 and 6). Comment: Hepatodiaphragmatic nodules can be seen in rats at any age and their occurrence in fetuses is considered presump- Hepatocytes contain a large well-defined single tive evidence of a congenital origin. While they appear to be rounded vacuole within each cell. protruding through the diaphragm and extending into the thor- Nucleus and cytoplasm displaced to the periphery. acic cavity, they actually are attached to and covered by a thin A few hepatocytes may contain one or more smaller fibrous portion of the diaphragm (Eustis et al. 1990). vacuoles. An incidence ranging from 1% to 11% has been reported for Microvesicular fatty change (Figure 7). hepatodiaphragmatic nodules in Fischer 344 rats (Eustis et al. 1990), with few cases reported in other rat stocks and strains. Hepatocytes partially or completely filled with Mice do not develop such nodules but may have focal lesions numerous small lipid vacuoles. similar to those in rat hepatodiaphragmatic nodules and with Affected hepatocytes may have a ‘‘foamy’’ appearance. large nuclei with large central nucleoli-like basophilic bodies. Small vacuoles do not normally displace the nucleus to the periphery in contrast to macrovesicular steatosis.

B. Hepatocellular Responses, Cellular Degeneration, Differential diagnosis: Injury, and Death Hydropic degeneration—clear cytoplasm without Introduction nuclear displacement. The function and structure of most liver cells are Glycogen accumulation—irregular and poorly defined relatively constrained by their genetic programs of metabolism, lacy clear spaces in the cytoplasm (rarefaction) usually differentiation, and specialization. While the cells of the hepatic with centrally located nuclei. parenchyma have the flexibility to adapt to changing physiological demands with reversible functional and morphological altera- Comment: There is a difference in preferred nomenclature tions, sufficient stress, or noxious stimuli may lead to inability to among pathologists for this change. Based strictly on an maintain homeostasis and adverse cellular adaptations. The mor- H&E-stained section, a diagnosis of cytoplasmic vacuolation phological response to injurious stimuli depends on the nature of of hepatocytes is a universally acceptable descriptive diagno- the injury and its severity and duration. Often at high doses, tar- sis. Based on the experience of the observer, the specific mor- geted cells go through a sequence of cellular degeneration fol- phological features of the cytoplasmic vacuolation may be lowed by cell death, but at lower doses degenerative changes sufficiently consistent with intracytoplasmic lipid accumula- do not necessarily lead to cell death. Consequentially, cellular tion to warrant a presumptive diagnosis of fatty change. The changes that do not lead to cell death or death of the animal may unequivocal demonstration of intracytoplasmic fat, however, be called ‘‘adaptive’’ changes that can be considered either requires a special stain. adverse or not adverse reactions, depending on the nature of the Fatty change can be induced by a number of different agents change. There are cellular adaptations involving metabolic or and is usually divided into two main types, namely, microvesi- functional alterations that lead to increases in cellular organelles cular and macrovesicular, although mixed forms can frequently and intracellular accumulations of a variety of endogenous and be observed (Greaves 2007; Gopinath, Prentice, and Lewis exogenous substances but allow the cell and animal to survive 1987; Goodman and Ishak 2006; Kanel and Korula 2005). and often live normally. Similar changes may occur in human Macrovesicular lipidosis is a reaction to a wide variety of liver, such as cholestasis, a common lesion in human liver injuries and can also be regarded as a physiological adapta- after long-term drug therapy. However, in animals, when the tiondemonstratedasanimbalancebetweenuptakeoflipids limits of adaptive responses are exceeded or do not occur in from blood and secretion of lipoproteins by the hepatocyte response to chemical exposure, irreversible cellular injury and (Goodman and Ishak 2006). Microvesicular lipidosis is usually cellular death occurs, with possible subsequent illness and death. Adaptive changes or doses of chemicals that induce 1 Electron microscopy or special staining needed for a definitive diagnosis.

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 10S THOOLEN ET AL. TOXICOLOGIC PATHOLOGY indicative of more serious hepatic dysfunction but can also Differential diagnosis: result from nutritional disturbances (Greaves 2007). Specific xenobiotics can induce either macrovesicular or Fatty change—round clear vacuoles tend to be single microvesicular lipidosis in humans (Kanel and Korula 2005). or multiple and discrete. In animal studies, it is common to see a mixture of macrovesi- Glycogen accumulation—irregular and poorly cular and microvesicular lipidosis. In those situations one can defined clear spaces in the cytoplasm (rarefaction) either diagnose the most prevalent form or record the findings usually with centrally located nuclei; positive stained as mixed. Commentary in the pathology narrative report might with periodic acid-Schiff staining. be appropriate, especially if recording the most prevalent form of lipidosis. Liver with admixed presence of glycogen and fatty Comment: Definitive diagnosis of phospholipidosis is not pos- change can be observed (Figures 8 and 9). sible based strictly on H&E-stained liver sections. A diagnosis Fatty change and necrosis may appear together although of cytoplasmic vacuolation of hepatocytes will typically be an they may differ in proportion. A number of causes other than acceptable descriptive diagnosis. Since the cytoplasmic xenobiotic exposure, such as chronic hepatic injury, diet, vacuolation may mimic microvesicular fatty change, a descrip- metabolic and hormonal status, debilitation of animals, and tive diagnosis of cytoplasmic vacuolation is recommended in fasting before necropsy, should be taken into consideration the absence of electron microscopy or special immunostaining. in reviewing these changes (Vollmar et al. 1999; Katoh and Phospholipidosis can be induced by xenobiotics with a Sugimoto 1982; Nagano et al. 2007; Denda et al. 2002). The cationic amphophilic structure (Halliwell 1997; Anderson and distribution can be either diffuse (e.g., ethionine) or zonal Borlak 2006; Reasor, Hastings, and Ulrich 2006; Chatman (e.g., centrilobular in CCl4; periportal in phosphorus toxicity; et al. 2009) (Figures 14 and 15). It is a lipid storage disorder midzonal in choline deficiency). Inadequate fixation proce- seen when complexes between xenobiotics and phospholipids dures may sometimes give rise to artifacts with microvesicu- accumulate within lysosomes. Phospholipidosis refers to a spe- lar vacuolation, although mostly with less clear cytoplasm cific form of hepatic vacuolation with the occurrence of con- (Li et al. 2003). centric membrane bound lysosomal myeloid bodies/lamellar Focal fatty change can sometimes be seen spontaneously and bodies that can be confirmed by specific staining and electron is usually described as such. A specific variation occurs near the microscopy (Hruban, Slesers, and Hopkins 1972; Obert et al. attachment of the falciform ligament and gallbladder in mice and 2007) (Figure 16). Definitive diagnosis requires electron is referred to as ‘‘tension lipidosis’’ (Harada et al. 1999) (Figures microscopy or positive immunostaining. Immunohistochem- 10 and 11). Spontaneous fatty change can differ between strains ical staining for a lysosomal-associated protein and adipophilin and is a normal finding in BALB mice. Livers of these mice are may be used to differentiate phospholipidosis from conven- typically paler than in other strains. Focal fatty change in the tional fatty change (Obert et al. 2007). Both preexisting neutral liver of rodents has previously been categorized as vacuolated fat and phospholipids can be observed in combination. The altered hepatic foci (Eustis et al. 1990), but current practice is macrovesicular and the microvesicular fatty change (vacuola- to diagnose this change as focal fatty change rather than as a tion) generally located at the cell periphery stains positively for focus of hepatic alteration (Figures 12 and 13). Oil Red-O and the membranes surrounding these lipid Fatty change can also be observed in combination with other vacuoles stain positively for adipophilin (a protein that forms hepatotoxic injuries (e.g., chronic liver toxicity, degeneration, the membrane around non-lysosomal lipid droplets) but neg- inflammation, and necrosis) or nutritional disturbance (e.g., ative for LAMP-2 (a lysosome-associated protein) by immu- diet, vitamin A excess) in both animals and man. Special stains nohistochemical techniques (Obert et al. 2007). This indicates on cryostat sections can demonstrate fat (e.g., Oil red O or that this vacuolation was due to accumulation of non- Sudan Black) (Jones 2002). lysosomal neutral lipid. Cytoplasmic microvesiculation located centrally in hepatocytes that exhibit positive immunohistochemical staining for LAMP-2 (Figure 17) but is nega- Phospholipidosis2 tive for Oil-Red-O and adipophilin is indicative of phospholipid accumulation (Obert et al. 2007). Synonym: Cytoplasmic vacuolation, foam cells. Amyloidosis (Figures 18 and 19) Pathogenesis: Induced by xenobiotics with a cationic amphophilic structure. Pathogenesis: Cellular process related to misfolding of protein. Diagnostic features: Diagnostic features:

Multiple irregular to round clear membrane-bound Deposition of pale, homogeneous, amorphous eosi- vacuoles. nophilic material. Tends to be a diffuse change affecting hepatocytes. Deposition often peri-sinusoidal, periportal, or involving blood vessel walls. 2 Electron microscopy or special staining needed for a definitive diagnosis. Localization is extracellular.

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Comment: This is a rare condition in rats but is a more com- Spencer et al. 1997; Yasui, Yase, and Ota 1991; DePass monage-relatedphenomenoninhamstersandmice et al. 1986). Mineralization can sometimes be observed in (Greaves 2007; BSTP 2007). The basis of the pathological combination with inflammation or neoplasia (Harada et al. change is the cell’s inability to prevent protein misfolding, 1999; Kanel and Karuda 2005). Mineral deposits can be to revert misfolded proteins to normal, or to eliminate mis- demonstrated by using additional stains (Alizarin Red, von folded proteins by degradation. This can result in deposition Kossa) (Churukian 2002). of potentially cytotoxic protein aggregates of amyloid as in other protein aggregation diseases (Aigelsreiter et al. 2007). The amyloid is predominantly composed of protein in a Pigmentation (Pigment Deposition) (Figures 21–25) beta-pleated sheet conformation. The incidence of spontaneous amyloidosis usually Pathogenesis: Incidental occurrence and secondary to cellular increaseswithageandiscommoninCD-1mice(Harada and erythryoid breakdown products; lipid peroxidation of cel- et al. 1996). Amyloid observed in the liver often is referred lular membranes; altered heme metabolism. to as secondary amyloidosis (serum amyloid A protein) and is seen in the sinusoids and within the portal vessel walls. Diagnostic features: Hepatocytes adjacent to sinusoidal amyloid deposits are often atrophic. A number of factors (e.g., species, age, strain, gen- Lipofuscin: der, endocrine status, diet, stress, and parasitism) can influence the occurrence of amyloidosis(Beregietal.1987;Coe Pigment can be seen in hepatocytes as well as in and Ross 1990; Lipman et al. 1993; Harada et al. 1996; Liu Kupffer cells. et al. 2007). Other organs are often involved in the deposition Mayvaryfrompaleyellowtodeepgranular of amyloid (e.g., kidney, nasal submucosa, lamina propria brown. intestines, heart, salivary gland, thyroid, adrenal cortex, lung, May be sudanophilic with autofluorescence under tongue, testis, ovary, and aorta). ultraviolet light. Amyloidosis can be confirmed with additional histo- Often located adjacent to bile canaliculi. chemical staining (Congo red) where it shows pink-red staining and apple green birefringence under polarized light (Vowles and Francis 2002; Kanel and Korula 2005) and by Iron/hemosiderin: immunohistochemistry. Can be yellow to brown. May be finely granular. Mineralization (Figure 20) Usually appears intracellularly in Kupffer cells and hepatocytes. Pathogenesis: Hypercalcemia secondary to diet or abnormal calcium metabolism; hepatocellular necrosis (dystrophic mineralization). Porphyrin:

Diagnostic features: Pigment is dense dark brown to red-brown and when viewed with polarization is bright red with a centrally Intra- or extracellular basophilic deposits, sometimes located dark ‘‘Maltese cross.’’ with calcification. Brilliant red fluorescence when viewed in fresh frozen sections; fades with exposure to ultravio- Differential diagnosis: let light. Most often located in bile ductules and bile Artifact—hematoxylin stain deposits in clear canaliculi. spaces. Pigment deposits—may be tinctorially different from Bile (cholestasis) (Figures 23–25): mineralization and often seen within macrophages. Intrabiliary accumulation of test compound or Appears as elongated pale green-brown plugs within metabolite. bile caniculi. May be associated with necrosis, inflammation, or Will appear in Kupffer cells following rupture of neoplasia. caniculi. Can appear as finely granular pigment within in hepa- Comment: Mineralization is rarely seen in the liver and gall- tocytes, which is common in human liver but much less bladder in rodents. Dietary factors (mineral content) and dis- common in rodents. turbance of calcium metabolism commonly influence the Not a common xenobiotic response in rodents; more process of hepatic mineralization (Harada et al. 1999; common in humans and monkeys.

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Differential diagnosis: (Ungar, Sullman, and Zuckerman 1976). Endogenous iron deposition can be found following breakdown of blood cells Artifact—hematoxylin stain deposits in clear spaces. (hemolytic event). Iron pigment can be found in Kupffer Formalin precipitated pigment—extracellular granu- cells, macrophages, and hepatocytes. In hepatocytes, the lar yellow-brown deposits often associated with iron is stored in the form of ferritin (ferric iron bound to erythrocytes. protein apoferritin) (Popp and Cattley 1991). A spontaneous Test compound/metabolite—may be distinctive for inherited predisposition for hepatic iron pigmentation has the specific compound. been reported in Sprague-Dawley rats (Masson and Roome Mineralization—basophilic deposits; may be associ- 1997), and iron deposition can be found in the aging mouse ated with calcification. liver (Harada et al. 1996). Iron can be demonstrated using Perls’ Prussian blue stain in which iron stains blue. Comment: A number of different pigments may be seen as an Hemosiderin slowly dissolves in acids, especially oxalic incidental finding within hepatocytes and Kupffer cells in acid. Non-aldehyde fixatives can remove hemosiderin or rodents. Some of them may increase and/or accumulate after alter it in such a way that reactions for iron are (false) neg- treatment. Definitive diagnosis of a specific pigment typically ative (Churukian 2002). Malarial pigment is seen in hepato- requires special stains. cytes and Kupffer cells of Plasmodium sp experimentally Lipofuscin or ceroid is sometimes referred to as ‘‘wear and infected mice. It is the pigment from the organism and not tear’’ or ‘‘aging’’ pigment and therefore is often observed in hemosiderin. older animals. It is considered to represent a breakdown of cell Porphyrin pigment normally occurs in tissues only in membranes. Lipofuscin accumulates in postmitotic and aging small amounts and is a precursor of the heme portion of cells. It has been shown to be a mixture of oxidized proteins and hemoglobin (Churukian 2002). Porphyrin deposition in the lipids, carbohydrates, and trace amount of metals (Seehafer and liver of rodents is found after administration of a number Pearce 2006). A variety of stimuli can accelerate the of compounds including griseofulvin where it can be seen accumulation of this pigment, such as drug and chemical expo- in association with hepatocellular neoplasia (Stejskal et al. sure, trauma and circulatory factors, and diet (Greaves 2007). 1975; Zatloukal et al. 2000; Knasmuller et al. 1997; Lipofuscin accumulation in the liver may be augmented by cer- Tschudy 1962). Griseofulvin administration in mice may tain chemicals (Kim and Kaminsky 1988; Marsman, 1995). result in inhibition of the mitochondrial enzyme ferrochela- Treatment of rats with PPAR alpha agonists such as fenofibrate tase and (compensatory) induction of ALA synthetase. and associated increased lipid peroxidation seen in rodents Griseofulvin-induced accumulation of porphyrins in mouse treated with hypolipidemic agents can induce lipofuscin accu- liver is followed by cell damage and necrotic and inflamma- mulation in liver after prolonged treatment (Nishimura et al. tory processes (Knasmuller et al. 1997). Proto-porphyrin 2007; Goel, Lalwani, and Reddy 1986; Reddy et al. 1982). pigment in liver of rats and mice is mainly found in the bile Increased lipofuscin accumulation has also been observed in par- ducts and leads to bile duct proliferation and portal inflam- tially hepatectomized liver of rats (Sigal et al. 1999). Lipofuscin mation, but can also occur in hepatocytes, Kupffer cells, and is insoluble in alcohols and xylene and other solvents normally portal macrophages (Hurst and Paget 1963). The birefrin- used in the preparation of slides. Special stains such as Smorl’s gence of porphyrin appears to be associated with bilamellar can be used to demonstrate the pigment. Storage granules appear components within the pigment (Stejskal et al. 1975). This gray with Sudan Black B, may be PAS-positive, and may stain pigment is also seen in combination with liver fibrosis and with Luxol fast blue and Ziehl-Neelsen (Jones 2002). cirrhosis, bile duct proliferation, periportal inflammation, Porphyrin pigment, a precursor of heme protein, is seen with and hepatocarcinogenesis (Kanel and Korula 2005; Hurst treatment of some xenobiotics. Bile pigment is a common find- and Paget 1963; Greaves 2007; Rank, Straka, and Bloomer ing when there is cholestasis secondary to obstruction of bile 1990). flow or when there is perturbation in bile metabolism. Bile pigment stains green with Hall’s method. Hemosiderin pigment represents precipitated iron that is Crystals (Figures 26–28) most frequently generated as a breakdown product of erythrocytes and is derived from hemoglobin and accumulates Pathogenesis: Hyperlipidemia (cholesterol crystals), Chi313 in the liver following local or systemic excess of iron. (Ym1) protein (eosinophilic biliary crystals). Deposition or iron may occur following excess dietary Diagnostic features: intake or treatment by xenobiotics (Popp and Cattley 1991; Greaves 2007; Travlos et al. 1996). Excess of iron Rhomboid or needle-like structures often birefringent following injection may be stored as hemosiderin and under polarized light. depositedinthereticuloendothelial component of the liver Needle-like crystals in the mouse can be intracellular (and other organs such as spleen and bone marrow) or extracellular and may be associated with intense (Bruguera 1999; Pitt et al. 1979). Intraperitoneal injection eosinophilic epithelial cytoplasm and extracellular of aflatoxin B1 can also induce hemosiderosis in hamsters crystals of various sizes.

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Differential diagnosis: Comment: Both intranuclear and intracytoplasmic inclusions are common findings in the aging mouse liver and Artifact—wispy blue hematoxylin deposits in clear may be seen in normal as well as neoplastic hepatocytes spaces. (Percy and Barthold 2001; Frith and Ward 1988; Irisarri and Hollander 1994). When the intranuclear inclusions represent invaginations of the cytoplasm into the nucleus, Comment: In hyperlipidemia, cholesterol crystals can deposit they may contain cytoplasmic organelles in electron micro- in the liver with or without granulomatous inflammation graphs (van Zwieten and Hollander 1997). Ultrastructu- (Greaves 2007; Graewin et al. 2004; Handley, Chien, and rally, three types of cytoplasmic inclusions have been Arbeeny 1983). During gall stone formation, in addition to described: dense reticulated substance in the dilated cister- classical rhomboid-shape monohydrate crystals, cholesterol nae of rough endoplasmic reticulum, fine granular sub- can also crystallize transiently as needle-, spiral-, and tubule- stance in rough endoplasmic reticulum, and non–membrane shaped crystals of anhydrous cholesterol (Dowling 2000). Eosi- bound dense granulofibrillar in the cytoplasm (Helyer and nophilic crystals have been described in intrahepatic bile ducts Petrelli 1978). and gallbladder of different laboratory mice strains, and some Kakizoe, Goldfarb, and Pugh (1989) have correlated the of these crystals have been shown to contain chitinase-like pro- incidences of cytoplasmic inclusion with hepatocellular teins confirmed by immunohistochemistry for Ym1 protein tumors in different mice strains. C57BL/6 mice are rela- (now Chi313) (Ward et al. 2001; Harbord et al. 2002). tively more resistant to hepatocarcinogens than C3H and Crystal formation may be associated with inflammatory C57BL/6 x C3H F1 mice. The tumors in the C57BL/6 mice and/or proliferative bile duct changes and fibrosis in mice and were unique in their early focal development of cells con- may also occur spontaneously (Lewis 1984; Rabstein, Peters, taining inclusions. The authors suggested that the higher and Spahn 1973; Enomoto et al. 1974). Numerous crystals can incidence of inclusions in liver might be related to slowing be demonstrated using a simple system of polarizing micro- of the tumor growth leading to lower incidence of hepato- scopy. Crystals are capable of producing plane-polarized light, cellular tumors in C57BL/6 mice. Other types of intracyto- thus showing birefringence. plasmic inclusions such as Mallory bodies, lamellated, and crystalloid inclusions have been described in mice treated with different chemicals and in lysosomal storage diseases Inclusions, Intranuclear, and Cytoplasmic (Figures 29–32) (Gebbia et al. 1985; Meierhenry et al. 1983; Rijhsinghani Synonyms: Inclusion bodies, intranuclear cytoplasmic invagi- et al. 1980; Shio et al. 1982). nation, acidophilic inclusions, globular bodies. Cytoplasmic vacuoles can occur in hepatocytes and endothelial cells in a postmortem time-dependent manner in fasted and non-fasted rats (Li et al. 2003). This artifact is espe- Pathogenesis: Protrusion of cytoplasm into an invagination of cially common in rats that are not exsanguinated at necropsy the hepatocyte nuclear membrane without the actual protrusion and the cytoplasmic vacuoles represent plasma influx into to necessarily being present in the plane of section. Seen in spe- affected cells (Figure 33). This artifact is more common in cific viral infections. Deposition of protein material within males than in females. hepatocyte cytoplasm. Diagnostic features: Hypertrophy, Hepatocellular (Figures 34–41) Intranuclear inclusions are round, distinct, usually Synonyms: Hepatocytomegaly. eccentrically located, and may partially or almost completely fill the nucleus. Pathogenesis: Metabolic enzyme induction causing increase in Contents of intranuclear inclusion bodies are often endoplasmic reticulum; increase in peroxisomes; increase in eosinophilic and may be granular or flocculent. mitochondria. Intracytoplasmic inclusions are round to oval, homo- genous, eosinophilic, and occur as single or multiple Diagnostic features: structures in the cytoplasm. Enlarged hepatocytes may be tinctorially distinct. Differential diagnosis: Cytoplasm may be homogeneous or granular. Zonal pattern of distribution (centrilobular, peripor- Enlarged nucleolus—one or more deeply basophilic tal, midzonal) may be present. structures in normal size nuclei. Involving most or all lobules. Viral inclusion bodies (cytomegalic virus, experi- Loss of hepatocellular plate architecture is possible. mental viral infections). Sinusoidal compression. Cytoplasmic vacuole artifact—postmortem plasma Concurrent degeneration and/or single cell necrosis is influx (Li et al. 2003). possible.

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Differential diagnosis: Diagnostic features:

Hepatocellular neoplasia—expansile mass with Decreased size of hepatocytes. altered growth pattern; loss of lobular structure. Small liver trabeculae with decreased cytoplasmic Regenerative hyperplastic nodules—altered growth volume, close proximity of hepatocyte nuclei, close pattern; distorted lobular pattern. proximity of portal tracts, and increased basophilia. Foci of cellular alteration—usually a discrete Hepatocyte nuclei may be smaller than normal. collection of cells within the hepatic parenchyma. May have a zonal distribution. Hepatocellular degeneration—affected cells May be associated with hepatocellular degeneration have increased cytoplasmic granularity and and/or single cell necrosis. eosinophilia. May be associated with increased sinusoidal size. Hepatocellular storage disease. Depletion of cytoplasmic glycogen. Polyploidy—enlarged nuclei and/or binuclear hepatocytes are often associated with increased cyto- Differential diagnosis: plasmic volume. Shrinkage artifact—retraction of softer tissue from Comment: Hepatocellular hypertrophy, secondary to increase firmer tissue. in microsomal enzymes often occurs with a zonal or specific Artifact of fixation or processing—poorly stained tis- lobular pattern and commonly occurs following exposure to sue with loss of normal structure. enzyme inducing xenobiotics. There is enlargement of the hepatocyte cytoplasm secondary to increase in the cytosolic Comment: Hepatocellular atrophy can be caused by a number of protein or number of organelles (e.g., smooth endoplasmic reti- factors such as inanition, starvation, hemodynamic changes, or culum, peroxisomes, mitochondria). Classically hepatocyte pressure atrophy from neoplasia (Yu et al. 1994; Gruttadauria hypertrophy occurs without increase in hepatocyte numbers et al. 2007; Belloni et al. 1988). Hepatocyte atrophy may be or DNA (i.e., hyperplasia or polyploidy), however, combina- associated with decrease in absolute liver weights in rats tions with increased mitoses do occur (e.g., PPAR-alpha (Belloni et al. 1988). Ultrastructurally atrophic hepatocytes agonists). have reduced amounts of glycogen and decreased numbers of Hepatocellular hypertrophy following enzyme induction mitochondria. is considered an adaptive response to chemical stress. Strain differences in responsiveness occur. While typically an adaptive response, excessive hypertrophy from enzyme Degeneration induction of hypertrophy can lead to hepatocellular degen- Introduction: In the diagnostic lexicon, degeneration is a non- eration and necrosis. Hepatocellular hypertrophy may be asso- specific diagnosis that provides limited useful information ciated with increase in absolute liver weights. Enzyme unless qualified to reflect the dominant morphological features. induction leading to hepatocellular hypertrophy may be It is often at the borderline between adaptation with resolution accompanied by some evidence of transient mitogenesis. back to normal structure and function and inability to adapt Hypertrophy that is panlobular may be difficult to appreciate leading to cell death. In human clinical medicine degenerative histologically because the contrast provided by a sublobular disease most often refers to chronic debility involving organs pattern is not evident. In some cases, hepatocyte hypertrophy and tissues that slowly accumulate damage over time. In rodent related to metabolic enzyme induction may not be evident to studies, degeneration may also be applicable to chronic the pathologist when liver weight increase is small for a group, debility, but more often it is used to reflect acute or chronic for example, less than 20%. cytological alterations with characteristic morphological fea- Hepatocellular enlargement or swelling may occur from tures. Combinations of different degenerative features may accumulation of glycogen, fat, or other substances and may occur with or without inflammation and/or necrosis. also be a feature of degeneration and some forms of hepa- Based on H&E-stained sections, distinction between tocellular necrosis. To avoid confusion with the more com- different forms of degeneration, hepatocellular hypertrophy mon usage of hepatocyte hypertrophy for physiological secondary to enzyme induction, other forms of hepatocellular enzyme induction, it is recommended that alternative forms enlargement such as glycogen accumulation/retention, and of hepatocyte enlargement not be diagnosed as hepatocellu- even early necrosis (a.k.a. onconosis) may be difficult. In some lar hypertrophy. cases special stains may be required to more clearly delineate the nature of the cytologic alteration. Based strictly on H&E staining, a descriptive diagnosis of cytoplasmic alteration is Hepatocellular Atrophy (Figures 42 and 43) recommended in lieu of interpretative diagnosis such as gran- Pathogenesis: Inanition, starvation, hemodynamic changes, or ular degeneration and hyaline degeneration. However, there are pressure atrophy from neoplasia. some degenerative lesions, such as hydropic degeneration and

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 15S cystic degeneration, that are more clearly established in tradi- formation (Gonzalez-Quintela et al. 2000; NTP Toxicology and tional pathology literature. The preferred diagnosis will be Caracinogenesis Studies Ethylene Glycol 1993; Peters et al. influenced by morphological features, conventional pathology 1983; Bruni 1960; Shea 1958; Omar, Elmesallamy, and Eassa practice, and the experience of the pathologist. 2005; Lin et al. 1996), but is rarely used as a separate description Glycogen accumulation in hepatocytes is a type of cytoplas- since a combination of findings is often present. Cytoplasmic mic alteration manifested on H&E-stained paraffin sections as alteration reflecting plasma influx is an artifact seen in non- clear spaces in the cytoplasm and a centrally located nucleus. exsanguinated rats in a postmortem time-dependent manner Intracellular accumulation of glycogen is a normal physiological (Li et al. 2003) (see Figure 33). response following food ingestion. Since rodents eat primarily in the evening hours, the largest amount of glycogen will be present Degeneration, Hydropic (Figure 45) during early morning hours. Intrahepatocyte glycogen is mobi- Synonyms: Cytoplasmic alteration, cytoplasmic change, hydro- lized throughout the day, initially being removed from centrilob- pic change, cloudy swelling. ular hepatocytes. Consequently the amount present varies depending upon whether the animals were fasted and on the time Pathogenesis: Intracytoplasmic fluid accumulation secondary of necropsy during the day. Failure to accumulate glycogen to disturbance of cell membrane integrity. because of inanition or abnormal glycogen retention may result from treatment-induced metabolic perturbations. Diagnostic features:

Cytoplasmic Alteration (Figure 44) Cytoplasmic vacuolation and ‘‘ballooning’’ with a centrally located nucleus. Synonyms: Cytoplasmic alteration, cytoplasmic change, granu- Lobular location may be centrilobular or periportal lar change, granular degeneration, hyaline degeneration, glyco- with increased clear cell change and cell swelling. gen accumulation; ground glass change. Differential diagnosis: Pathogenesis: Often xenobiotic-induced and may be associated with other forms of liver damage. Cytoplasmic vacuole artifact—postmortem plasma Diagnostic features: influx. Glycogen accumulation—hepatocytes not markedly Affected cells may show increased cytoplasmic gran- enlarged; cytoplasmic clear areas are irregular. ularity, cell swelling, and eosinophilia. Comment: Because of disturbance of the cell membrane integ- Differential diagnosis: rity, accumulation of intracytoplasmic fluid may occur. This causes vacuolation and ‘‘ballooning’’ of cells. This change can Artifact of fixation or processing—poorly stained be caused by a number of xenobiotics with differing lobular tissue with loss of normal structure. localization and may be a precursor to hepatocyte necrosis Hepatocyte hypertrophy—cytoplasmic volume (Gkretsi et al. 2007; Wang et al. 2007; Peichoto et al. 2006; increased with uniform finely granular texture; usu- Matsumoto et al. 2006; Chengelis 1988). ally associated with microsomal enzyme induction or peroxisome proliferation. Degeneration, Cystic (Figure 46 and 47) Cytoplasmic vacuole artifact—postmortem plasma Synonyms: Spongiosis hepatis (traditional diagnostic term pre- influx. ferred by many pathologists). Coagulation necrosis—loss of cytoplasmic and nuclear detail. Pathogenesis: Cystic enlargement of perisinusoidal stellate cells (Ito cells) particularly observed in aging rats. Comment: What has been described as granular degeneration can be seen in combination with other forms of liver damage Diagnostic features: (e.g., necrosis, hydropic degeneration, inflammation) (Huang et al. 2007; Gokalp et al. 2003; Datta et al. 1998; Xu et al. Multi-loculated cyst(s) lined by fine septa containing 1992; Aydin et al. 2003). Hepatocellular granularity may be fine flocculent eosinophilic material (PAS-positive). due to swelling of cell organelles or increase in the numbers The cysts are not lined by endothelial cells and do not of cell organelles including peroxisomes, mitochondria, and compress the surrounding liver parenchyma. smooth endoplasmic reticulum. Some pathologists do not con- May be accompanied by occasional erythrocytes or sider granular degeneration to be a distinct entity and do not leukocytes. include it in their diagnostic lexicon. May be observed within altered hepatic foci and liver Hyaline degeneration has been described by a number of tumors. authors, sometimes in combination with Mallory body Affected cells may be markedly enlarged.

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Differential diagnosis: Single Cell Necrosis (Apoptosis) (Figures 48–50) Diagnostic features: Angiectasis (Peliosis hepatis; vascular ectasia)— dilated endothelial lined vascular spaces that often Affected hepatocytes may have condensed hyper- contain blood cells. eosinophilic cytoplasm and a somewhat angular outline. Sinusoidal dilatation—sinusoidal structure evident Not associated with an inflammatory response unless and spaces lined by endothelial cells. there is simultaneous necrosis. Necrosis—loss of nuclear and cytoplasmic detail and May occur spontaneously with one or two affected loss of stain affinity. hepatocytes present in an occasional hepatic lobule. Hemangioma—expansile structure lined by flattened May be exacerbated by treatment. endothelial cells; may be associated with parenchy- In standard H&E-stained sections, apoptotic hepato- mal compression. cytes (apoptotic bodies) are usually rounded with Hemangiosarcoma—expansile mass lined by plump condensed cytoplasm. endothelial cells and/or layers of endothelial cells Rounded apoptotic bodies are typically surrounded and associated with destruction of hepatic by a clear halo. parenchyma. Fragments of nuclear material may be present within affected cells. Comment: Spontaneous and xenobiotic-induced cystic degen- Apoptotic bodies are frequently phagocytosized by eration/spongiosis hepatis may occur in rats (Karbe and Kerlin adjacent normal cells including hepatocytes and 2002; Bannasch 2003; Babich et al. 2004; Newton et al. 2001). macrophages. It is more common in aging rats with some male predilection. It is less common in mice. This lesion may be seen in or associ- Differential diagnosis: ated with other hepatic lesions (necrosis, regeneration; foci of cellular alteration; hepatocellular neoplasms). The pathogen- Small foci of necrosis—typically cells are swollen esis is not fully understood (Bannasch, Block, and Zerban and there is loss of membrane integrity; usually not 1981; Karbe and Kerlin 2002). rounded; less intensely stained than apoptotic bodies; may be accompanied by inflammatory cells.

Cell Death (Necrosis, Apoptosis) Comment: Apoptosis is a form of genetically controlled Introduction: In the fully developed organism, cell death is the ‘‘programmed cell death.’’ Microscopically in H&E- ultimate result of irreversible cellular injury. Cellular death in stained tissue sections, apoptosis appears as dense eosino- the liver is manifested by a spectrum of morphological pat- philic shrunken cell bodies with maintenance of membrane terns that can occur alone or in combinations. However, there integrity, nuclear fragmentation and cytoplasmic budding, are two primary manifestations of cell death: necrosis and and without an inflammatory response. Definitive diagnosis apoptosis. For decades a form of necrosis involving individual of apoptosis can be made by histological findings and isolated hepatocytes has been diagnosed as ‘‘single cell necro- confirmed by distinctive electron microscopic features. Con- sis.’’ This particular change is now regarded as ‘‘apoptosis’’ sequently, use of a diagnosis of ‘‘single cell necrosis’’ is by most pathologists (Levin 1999; Levin et al. 1999; Elmore appropriate based strictly on H&E staining. The use of 2007) when the majority of injured cells have the typical TUNEL kits or caspase immunostaining may assist in diag- apoptotic morphology. Provided that there is no accompany- nosing apoptosis and enumerating affected cells, but necro- ing inflammatory reaction, the two terms are synonymous. sis may also be immunopositive. Inhibition of apoptosis However, since a diagnosis of ‘‘apoptosis’’ implies a specific also plays a key role in the process of carcinogenesis (Fos- sequence of biochemical and morphological events and ter 2000). Although apoptosis can be observed sponta- should ideally be supported by electron microscopy, it may neously in the liver, certain chemicals may be able to be more prudent to diagnose single cell necrosis unless there trigger direct stimulation of pro-apoptotic pathways in hepa- is definitive proof of apoptosis provided by electron micro- tocytes (Feldmann 1997; Reed 1998). Apoptosis can also scopy (Levin et al. 1999). It can be mentioned in the pathol- accompany treatment-related zonal necrosis in the liver, ogy narrative that the observed ‘‘single cell necrosis’’ is especially in situations wheretheremaybeaxenobiotic- morphologically consistent with ‘‘apoptosis.’’ induced effect (Cullen 2005; Greaves et al. 2001). While apoptosis represents a specific genetically programmed form of cell death unaccompanied by an inflamma- Pathogenesis: Direct or indirect cellular damage, including tory response, there are situations where small numbers of anoxia. Apoptosis (a.k.a., single cell necrosis) can occur spon- cells and even occasional single cells characterized by cell taneously in liver and may also be exacerbated or induced by swelling can undergo necrosis without an inflammatory treatment. response. This represents an early stage of conventional

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 17S necrosis, may occur within hours after exposure to a xenobio- seen after anoxia, or exposure to tannic acid, chloroform, or tic, and should not be diagnosed as single cell necrosis (apop- other hepatotoxic agents (Gopinath, Prentice, and Lewis tosis). A more appropriate diagnosis for this situation is focal 1987). This zone (Rappaport zone 3) is particularly vulnerable necrosis (see the following). to ischemic damage because of its low oxygen gradient and generation of toxic metabolites due to high content of xenobio- Necrosis, Focal/Multifocal (Figures 51–53) tic metabolizing enzymes (Comporti 1985; Walker, Racz, and McElligott 1985). Diagnostic features: Diagnostic features: Single or multiple foci of a few pale staining hepatocytes. Early necrotic hepatocytes are swollen. Usually retain normal morphological outline. Cytoplasm has increased eosinophilia. May be associated with inflammation. Nucleus undergoing lysis, not pyknosis. May have an irregular distribution but can also occur in May have a minimal associated inflammatory the subcapsular areas with minimal or no inflammation. reaction. Early lesions typically consist of three or four hepa- Can be accompanied by glycogen depletion, hydro- tocytes, but as the lesions progress more hepatocytes pic degeneration, fatty change, hemorrhage, and may be involved. ‘‘ballooning’’ of hepatocytes. Subcapsular necrosis may sometimes be observed in combination with hypertrophy. Midzonal (Figures 60–61)

Differential diagnosis: This necrosis is the least common form of zonal necrosis and is mediated by specific toxicants (e.g., furan, concavalin-A, beryllium) (Wilson et al. 1992; Boyd 1981; Seawright 1972; Foci of extramedullary hematopoiesis—mature and/ or immature erythroid and myeloid cell aggregates Satoh et al. 1996; Cheng 1956). The location is considered without accompanying hepatocyte necrosis. specific and has a metabolic basis. Foci of inflammatory cell infiltrate—aggregates of Diagnostic features: cells, usually mononuclear cells, in absence of obvious hepatocellular necrosis. Seen as a band of swollen and eosinophilic cells Infectious disease (MHV, Ectromelia, Clostridium intermediate to the central vein (zone III) and the por- pilliforme, Helicobacter hepaticus, Parvo virus, tal triad (zone I). Noro virus)—a spectrum of acute to chronic active Nucleus undergoing lysis. inflammation, degenerative, and proliferative changes Two to three cells in thickness in the middle of the specific for the infectious disease entity. lobule.

Comment: Some pathologists use focal for both focal and mul- Periportal (Figure 62) tifocal, referring to the nature of the lesion rather than its actual distribution. A severity grade can be used to reflect the multi- Hepatic necrosis in the periportal zone is observed follow- focal nature of the lesion. Focal, multifocal, and subcapsular ing a variety of agents (e.g., phosphorus, ferrous sulphate, necrosis is occasionally seen in untreated rodents and may be allyl alcohol) (Kanel and Korula 2005; Atzori and Congiu a terminal event potentially due to hypoxic change secondary 1996; Sasse and Maly 1991). Affected cells may encircle the to impaired blood flow. Subcapsular necrosis has also been portal tract (Popp and Cattley 1991) and may be associated reported from direct pressure secondary to gastric distention with inflammatory and other changes (Ward, Anver, et al. and from some types of restraint (Parker and Gibson 1995; 1994; Ward, Fox, et al. 1994; NTP Technical Report on the Nyska et al. 1992) Toxicology and Carcinogenesis Studies of a Binary Mixture 2006; NTP Technical Report on the Toxicology and Carcino- Necrosis, Zonal (Centrilobular, Midzonal, Periportal, genesis Studies of 2, 3, 7, 8-tetracholorodibenzo-p-dioxin Diffuse) 2006). Pathogenesis: Secondary to direct or indirect damage from Diagnostic features: xenobiotic exposure; tissue anoxia. Swollen and/or eosinophilic hepatocytes may completely encircle the portal tract. Centrilobular (Figures 54–59) Nucleus undergoing lysis. Sometimes referred to as periacinar necrosis, it consists of May be accompanied by periportal inflammation, fibro- irreversible cell death of centrilobular hepatocytes and is often sis, bile duct proliferation, and oval cell hyperplasia.

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Diffuse (Figures 63 and 64) Pathogenesis: Duplication of nuclear material in absence of cytokinesis. Variations in nuclear size and ploidy (karyome- Synonym: Massive necrosis, panlobular necrosis. galy and/or anisokaryosis) are common in aging rodents. Diagnostic features: Diagnostic features: Necrosis involving a large portion of a liver lobe. Hepatocytes with either two or more nuclei or with a May be associated with torsion of a liver lobe. single enlarged nucleus which may be tetraploid or May be randomly distributed throughout the liver octaploid. without a specific lobular localization. Polyploid hepatocytes are frequently larger than adjacent diploid hepatocytes. Differential diagnosis: Anisokaryosis is randomly distributed in the hepatic lobule with more affected hepatocytes in the centri- Autolysis—loss of microscopic tissue structure and lobular region. stain affinity; pale eosinophilic staining and absence of nuclear detail. Differential diagnosis: Torsion of a liver lobe—Affects an entire liver lobe, loss of microscopic structure. Hepatocellular neoplasia—mass or expansile prolif- Infarction—usually an angularly shaped wedge or eration of hepatocytes with distortion or loss of lobu- area of tissue necrosis; may be associated with a lar architecture. nearby thrombus. Hepatocellular hypertrophy (enzyme induction)— increase in cytoplasmic volume not typically associ- Comment: Zonal necrosis is typically associated with expo- ated with increased nuclear size or number. sure to xenobiotics that either directly damage hepatocytes or cause damage following metabolic activation by endogenous or induced enzymes. There is often a concentration Comment: Karyocytomegaly is a reflection of hepatocyte gradient within the hepatic lobule with more extensive lob- polyploidy that occurs when there is duplication of nuclear ular involvement associated with higher doses of the toxic material in the absence of cytokinesis. The result is an increase agent. in the number of diploid nuclei per hepatocyte or an increase in Hepatocellular necrosis can occur spontaneously in rodents the ploidy level of a single hepatocyte nucleus. Polyploidy or be induced by xenobiotics, toxins, or following treatment at increases with age in some strains of mice as well as following high dosages with associated tissue anoxia, circulatory some treatment regimens resulting in hepatocytomegaly as derangements, and biliary stasis. Necrosis (centrilobular, mid- well as karyomegaly (Harada et al. 1996). Variations in cell zonal, periportal) might be accompanied by other histological size as well as in nuclei and polyploidy are also common in changes (fatty change, congestion, hemorrhage, inflammation, aging rats of different strains. Karyomegaly and anisokaryosis bile stasis, fibrosis, etc.) to form a myriad of pathological are normal incidental findings, especially in older mice (Percy changes. The distribution might also cross certain zones and and Barthold 2001). Increase in cell size (cytomegaly) may may manifest as ‘‘bridging necrosis’’ showing confluence of accompany the increase in hepatocyte ploidy. Anisokaryosis the lesions (e.g., central to central veins, portal tract to portal (inequality in size of nuclei) is more common and dramatic tract, or portal tract to central zones). Bridging necrosis may in mice than in rats. ultimately give rise to bridging fibrosis. The development of polyploidy and its pattern vary among A specific form of necrosis, ‘‘piecemeal necrosis,’’ is char- strains. C3H and DBA mice more commonly have octaploid acterized by necrosis of the limiting plate of the portal tract at cells with two tetraploid nuclei in adult liver while NZB and the the interface of hepatocytes and connective tissue of the portal out-bred strain NMRI at the corresponding age show a higher tract, usually accompanied by inflammation, can be immune- proportion of diploid cells with strikingly low proportions of mediated, and is seen in mice with resemblance to chronic tetraploid cells. Polyploidy has been observed in the early life active hepatitis in man (Kitamura et al. 1992; Nonomura (three weeks) in Ercc1 null mice. This premature polyploidy in et al. 1991; Kuriki et al. 1983). Ercc1-deficient liver is most likely caused by increased levels of p21 in response to accumulating DNA damage (Chipchase, O’Neill, and Melton 2003). Toxic injury caused by chemicals Karyocytomegaly and/or Multinucleated Hepatocytes such as phenobarbitone (Martin et al. 2001) and partial hepa- (Figures 65 and 66) tectomy also induce an increase in ploidy, usually associated Synonyms: Karyocytomegaly, multinucleated hepato- with extensive but transient hepatocyte proliferation (Gerlyng cytes, binucleated hepatocytes, karyomegaly, nuclear et al. 1993). hypertrophy, hepatocytomegaly, polyploidy, anisonucleosis, Although anisonucleosis (polyploidy) is known to occur as anisokaryosis. an age-related phenomenon, the nuclear and cellular changes

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 19S can also be induced by xenobiotics (Schoental and Magee are often referred to as simple or multiloculated biliary cysts 1959; Jones and Butler 1975; Singh et al. 2007; Nyska et al. (Goodman et al. 1994). Polycystic liver can be observed in 2002; Guzman and Solter 2002; Lalwani et al. 1997; Travlos the rat (Muff et al. 2006; Sato et al. 2006) and hamster (Percy et al. 1996; Kari et al. 1995; Herman et al. 2002). In addition, and Barthold 2001), resembling Caroli’s disease in humans multinucleated cells (formed by cell fusion rather than divi- (Clemens et al. 1980; Numan et al. 1986; Serra, Recalde, and sion) can be formed in rats after administration of 2, 3, 7, 8- Martellotto 1987). The cysts seen in polycystic disease are mul- tetrachloro-dibenzo-p-dioxin (Gopinath, Prentice, and Lewis tiple and seen diffusely throughout the liver and are of variable 1987; Jones and Butler 1975). Eosinophilic cytoplasmic inclu- size but generally large compared to the smaller biliary cysts. sions may be seen in affected hepatocyte nuclei because of cell membrane invaginations. C. Inflammatory Cell Infiltrates and Hepatic Inflammation Cysts, Biliary (Hepatic Cysts) (Figures 67–69) (Hepatitis) Pathogenesis: More common in aging animals occurs as a Introduction dilation of biliary structures. A variety of focal, multifocal, and more generalized infiltra- Diagnostic features: tions of inflammatory cells are frequently present in liver tissue. Changes range from acute inflammatory cell infiltrate(s) Range in size from small to very large. or occasional aggregates of lymphocytes/lymphohistiocytic Single or multiple cysts. cells/foci of mononuclear cells without associated alterations Macroscopically may contain clear to pale yellow of adjacent hepatocytes, to large panlobular patches of distinct fluid. hepatocyte necrosis accompanied by polymorphonuclear and May occur anywhere in the liver and may be unilocu- mononuclear (lymphocytes, plasma cells, macrophages) cellular or multilocular. lar infiltrates. ‘‘Mononuclear cell’’ can be used when there is a Multilocular cysts are divided into variably sized mixture of cell types (lymphocytes; less often macrophages and compartments by partial or complete connective plasma cells) or the cell type is mononuclear but cannot be tissue septa. unequivocally identified in H&E stain. If a cell type predomi- Cyst walls are characteristically lined by flattened to nates, then the infiltrate should be classified as lymphocytic, cuboidal epithelium. plasmacytic, or histiocytic. While etiological agents (e.g., bac- May be mild compression of adjacent hepatic teria, virus, parasite) may be present, in most safety assessment parenchyma. studies the causes of significant inflammation are either cryptic or are attributed to a specific treatment regimen. Inflammatory Differential diagnosis: reactions in the liver may be accompanied by oval cell and fibroblast proliferation and the propensity for hepatocellular Cystic degeneration—consists of markedly enlarged proliferative responses to replace lost parenchyma. cells with finely flocculent pale eosinophilic cytoplasm. It is recommended that use of the diagnostic term Angiectasis (Peliosis hepatis)—dilated vascular spaces ‘‘inflammation’’ for the liver should be used sparingly. Liver lined by endothelial cells; may contain blood cells. inflammation (hepatitis) is operationally defined as a constella- Bile duct dilation—dilated bile ducts lined by cuboi- tion of changes that represent a severe and generalized liver dal epithelium; not multiloculated. reaction and would require multiple diagnostic terms to ade- Parasitic cyst—may have thickened wall and contain quately characterize (Figure 70). This type of reaction is not parasite tissue. typically encountered in conventional rodent toxicity studies. Cholangioma—may cause compression of adjacent Traditionally, hepatic inflammatory responses have been parenchyma and spaces lined by more endothelial classified as acute, subacute, chronic, granulomatous, and so cells than in biliary cysts. on. These terms are somewhat interpretative, lack precise def- initions, vary depending upon study duration, usually do not Comment: Biliary cysts are commonly seen in older rats (Burek consist of a singular cell type, and do not have exclusive 1978; Greaves 2007; Harada et al. 1999). Solitary cysts can be pathognomic features. A more descriptive approach is recom- observed without major adjacent morphology changes of the sur- mended and can be qualified by lesion distribution or use of rounding tissue. However, depending on the location, adjacent subclassification and discretionary qualifiers (Figure 71). parenchyma may contain pressure atrophy of the hepatic cords of the liver, fibrosis, hemosiderin deposition, proliferation of bile Infiltration, Inflammatory Cell ducts, or periportal lymphocytic infiltration. Single cysts are often caused by cystic dilatation of the intrahepatic bile ducts Pathogenesis: Infiltration of different inflammatory cells is (Sato et al. 2005). Multiple cysts are observed also in hepatic typically a response to parenchymal cell death with causes polycystic disease, where they occur alone or in combination ranging from infectious agents, exposure to toxicants, genera- with polycystic kidney disease (Masyuk et al. 2004, 2007). They tion of toxic metabolites, and tissue anoxia.

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Infiltration, Neutrophil (Figure 72) Infiltration, Mononuclear (Figures 73–79) Synonyms: Inflammation, acute; acute inflammatory cell infil- Synonyms: Inflammation, chronic; mononuclear cell aggregates; trate(s), focus/foci of acute inflammatory cells; aggregate(s) of inflammation, granulomatous, focus/foci of mononuclear cells. acute inflammatory cells. Pathogenesis: Persistent noxious stimuli associated with infec- Diagnostic features: tion, toxic xenobiotics, continued low level parenchymal cell death, and immune mediated effects. Predominantly neutrophilic (and in some specific Specific subtypes may include: infiltration, lymphocytes; types of experiments, eosinophilic) cells present as infiltration, histiocytes (monocytes); infiltration, plasma cells. focal aggregates often associated with dying hepatocytes or at the periphery of large areas of hepatocyte Diagnostic features: necrosis. A few lymphocytes and occasional macrophages may be present. Infiltrating cells include lymphocytes, plasma cells, Infiltrating cells are usually focal or multifocal. macrophages, and occasionally multinucleated giant Necrosis of scattered individual cells or small clus- cells. ters of cells without associated infiltrating neutro- Infiltrating cells may be focal, multifocal, or diffuse phils may also be present in some areas. in distribution. Can be associated with whole regions of contiguous Randomly distributed aggregates of mononuclear affected lobules and confluent hepatic necrosis cells (primarily lymphocytes) without any accompa- extending between adjacent lobules (bridging hepatic nying hepatocyte degeneration or necrosis may be necrosis). common in older animals. Hemorrhage may be associated with larger lesions. Periportal aggregates of predominantly lymphoid Oval cell proliferation may be present. cells in portal areas may be present. Evidence of liver cell necrosis may be minimal to Differential diagnosis: mild with degeneration of scattered cells. Mononuclear cells with disruption of the limiting Foci of extramedullary hematopoiesis—mature and/ plate often infiltrate portal tracts. or immature erythroid and myeloid cell aggregates Bile duct hyperplasia may be present in some portal without accompanying hepatocyte necrosis. areas. Granulocytic leukemia—hepatic parenchyma infil- An increase in mononuclear cells within sinusoids trated and replaced by a monomorphic population may be seen. of neutrophils or neutrophil precursors. Some capsular fibrosis and periportal fibrosis may be Chronic inflammation—inflammatory cells include present. primarily mononuclear cell (largely lymphocytes, There may be evidence of hepatocellular regeneration. macrophages) and may include fibrosis and oval cell hyperplasia. Differential diagnosis:

Histiocytic sarcoma—hepatic parenchyma infiltrated Comment: While neutrophilic infiltration in the liver is and replaced by collections of histiocytic cells that primarily a response to liver cell injury and necrosis, a few can be pleomorphic. lymphocytes or macrophages may also be present. In addi- Lymphoma—hepatic parenchyma infiltrated and tion, foci of neutrophils without apparent hepatocyte necrosis replaced by a monomorphic population of lymphoid may be present, especially in situations of a transient effect cells. on the liver. In florid reactions, areas of necrosis include Early lymphoma may mimic age-associated focal degenerating neutrophils admixed with the necrotic hepatic aggregates of lymphoid cells. parenchymal cells. Foci of extramedullary hematopoiesis—mature and/or The extent of parenchymal cell death eliciting inflamma- immature erythroid and myeloid cell aggregates with- tory cell infiltration varies from minimal microfocal lesions out accompanying hepatocyte necrosis. to large patches of coagulation necrosis encompassing multi- Viral hepatitis—may be difficult to distinguish from ple contiguous lobules. For xenobiotic-induced cell death and chronic (active) inflammation; evidence of a viral inflammation, the severity of the lesions is often a function of etiology can be supportive. the dose of the hepatotoxicant. Apoptotic cell death may occur along with conventional necrosis. Depending upon Comment: Mononuclear cell infiltration spans a wide spec- etiology, acute inflammation can have a specific lobular dis- trum of morphological features and severities depending upon tribution with the possibility of portal or centilobular bridging the extent and duration of liver damage and any ongoing between adjacent lobules. regenerative response. In distinction from acute inflammation,

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 21S there are typically fewer neutrophils seen in chronic lesions and system is active. Others may consider chronic active inflamma- there is more involvement of portal areas with infiltrating mono- tion simply as a form of chronic inflammation with areas of nuclear cells and disruption of limiting plates. Granulomatous neutrophilic infiltration in the inflammatory process and inflammation with characteristic focal or multifocal nodular col- prefer to address that in their accompanying pathology narra- lections of mononuclear epitheloid cells and occasional giant tive. A combined neutrophilic and mononuclear infiltrate cells associated with areas of hepatocyte necrosis can be consid- (chronic active inflammation) is a common response found in ered a specific subtype of mononuclear cells infiltration and part mice chronically infected with Helicobacter hepaticus. of the spectrum comprising chronic inflammation of the liver (Figures 74–77). Some pathologists consider granulomatous Infiltration, Peribiliary (Intrahepatic) (Figure 81) inflammation of the liver to be a form of chronic inflammation Pathogenesis: Age associated change that may be exacerbated when there is a prolonged duration with predominant presence of by treatment. epitheloid cells/macrophages and prefer to address the fact that there may be areas containing mononuclear epitheloid cells and Diagnostic features: multinucleated giant cells in the chronic inflammatory process in an accompanying narrative. Fatty material and cholesterol Peribiliary aggregates of inflammatory cells affecting deposits may accumulate in some granulomatous inflammatory a few to many portal areas. reactions. Such localized accumulations of abundant cholesterol Sometimes accompanied by fibrosis. have been referred to as cholesterol granulomas (Figures 78 and May be associated with some degree of bile duct 79). hyperplasia. When only few isolated collections of mononuclear cells are Differential diagnosis: present, some pathologists may diagnose them as focal mononuclear cell aggregates. These focal accumulations are considered Cholangiofibrosis—proliferative and metaplastic by some to be a background lesion, and for these aggregates biliary response plus fibrosis extending into the hepa- using the cell type in the diagnosis, instead of inflammation or tic parenchyma; may be subcapsular. inflammatory cell infiltrate, may be preferable and less mislead- ing. The frequency of these mononuclear cell aggregates may Comment: A minimal to moderate peribiliary inflammatory be exacerbated by treatment. Helicobacter sp. and murine noro- cell infiltration consisting primarily of mononuclear cells can virus infections in mice may cause these incidental lesions. occur commonly in the livers of rats and mice and increases in incidence with animal age. Persistent obstruction to biliary Infiltration, Mixed (Figure 80) flow may also lead to bile duct inflammation in hepatic portal Synonym: Infiltration, purulent, and mononuclear; chronic areas. Although this background lesion may be considered a active inflammation; mixed inflammatory cell focus/foci. subtype of mononuclear cell infiltration (see previous), it is fre- Diagnostic features: quently diagnosed separately when exacerbated by treatment. Fibrosis (Figures 82 and 83) Infiltration of mononuclear cells along with neutrophils. Pathogenesis: A reaction to acute or prolonged hepatotoxicity. Areas of active hepatocellular degeneration and/or Diagnostic features: necrosis often present. Evidence of hepatocellular regeneration may be present. The presence of connective tissue in the liver above Intralobular distribution is often random. the normal low rate seen in portal areas. May have diagnostic features common for both neutro- Peribiliary fibrosis particularly in aged rats. philic and mononuclear infiltration (see previous). Three patterns of fibrosis are seen in the rodent liver: Differential diagnosis: pericellular, peribiliary, and postnecrotic. The pericellular pattern is most common in mice. Histiocytic sarcoma—hepatic parenchyma infiltrated Normal hepatic architecture maintained. and replaced by collections of histiocytic cells that Increased prominence of fibrosis, which may sur- can be pleomorphic. round hepatic lobules and bridge between adjacent Lymphoma—hepatic parenchyma infiltrated and portal areas. replaced by a monomorphic population of lymphoid Hyperplastic nodules of hepatocytes separated by cells. septae of connective tissue can be seen in rats but is less common in mice. Comment: Some pathologists consider a combined neutrophilic Oval cell proliferation often spreading from peripor- and mononuclear inflammatory cell infiltration as chronic tal areas. active inflammation. This type of response is suggestive that Masson trichrome, van Gieson, or Silver stains can be the adverse stimulus is still present and/or that the immune used to delineate fibrosis.

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Differential diagnosis: Diagnostic features:

Cholangiofibrosis—marked pericholangial fibrosis Focal to multifocal areas of necrosis with or without with proliferation of biliary epithelium and metaplas- inflammation. tic changes of glandular epithelium (e.g., goblet cells, Silver stain positive helical to rod-shaped bacteria in Paneth cells). or adjacent to the lesions, often can be seen between Regenerative hyperplasia—nodular growth pattern; hepatocytes (because organisms are in the bile distorted lobular pattern. caniculi). Multiple foci of cellular alteration—discrete collec- Chronic lesions—focal, multifocal, to diffuse may tions of cells within the hepatic parenchyma. include inflammatory cell foci, hepatocytomegaly, Multiple hepatocellular adenomas. oval cell hyperplasia, and cholangitis. Scirrhous carcinoma—neoplastic epithelial cells will Oval cell hyperplasia, focal to diffuse, minimal to be embedded in a collagenous connective tissue pro- marked. liferative response. Differential diagnosis: Comment: The pattern of fibrotic response to chronic injury varies among the species. When hepatic fibrosis is Inflammation, focal or multifocal, acute or chronic, accompanied by a nodular or non-nodular regenerative unknown etiology. response in the liver, it may be considered by some to repre- Murine norovirus infection. sent cirrhosis. Classical cirrhosis is rare in rodents in contrast Tyzzer’s disease. to dogs and humans and cannot reliably or consistently separate from a robust fibrotic reaction with associated nodular regeneration, oval cell proliferation, and bile duct hyperpla- Comment: AnumberofdifferentHelicobacter spp. have been sia. Such a robust fibrosis can be induced in rodents with pro- identified that can affect the rodent liver spontaneously longed or repeated exposure to certain chemicals (carbon (Ward, Anver, et al. 1994; Ward, Fox, et al. 1994; Goto tetrachloride, alcohol, tetrachlorovinphos, diallylphthalate), et al. 2000; Zenner 1999). Helicobacter spp. can cause an dietary lipotrope deficiency, or chronic hepatitis secondary increase in hepatocellular tumors in certain strains of infected to persistent infection (Ward 1997). We see no advantage mice, but are also known to generate liver lesions (Ward, Fox, in calling severe hepatic reaction cirrhosis in contrast to a et al. 1994; Tian et al. 2005; Rogers and Fox 2004) and can diagnosis of hepatic fibrosis with an appropriate severity promote experimental carcinogenesis of the liver (NTP Tox- grade. The specific morphological features of this response icology and Carcinogenesis Studies of Theophylline 1998; can easily be addressed in the pathology narrative. It should Zenner 1999; Diwan et al. 1997) in rodents. The pathogenicity be noted that hepatocellular neoplasms might arise in severe of the bacteria can vary with strain of bacteria and mouse hepatic fibrosis. strain, stock or line. Helicobacter hepaticus may cause acute to subchronic minimal to severe lesions in livers of susceptible mice such D. Infectious Diseases as A strain, C3H, and BALB/c (Ward, Anver, et al. 1994). Introduction Many mouse strains are resistant to liver infection but A strain mice are the most susceptible (Ward, Anver, et al. Infectious diseases of the mouse liver are an important 1994; Ward, Fox, et al. 1994). Incidental findings of focal group of conditions that may interfere with toxicology and or multifocal necrosis in the liver with or without inflamma- carcinogenesis studies. The histological changes associated tory cells such as macrophages, lymphocytes, and neutro- with the diseases described in the following may be recorded phils can be seen. In severely affected livers, more severe using the nomenclature described previously under inflamma- chronic lesions can be observed. H. hepaticus hepatic tion and inflammatory cell infiltrates but they are also pre- lesions are more common in males than females and inci- sented here as a separate category of disease diagnoses to dence is increased in mice six months of age or older (Percy help pathologists diagnose the infections, which can be con- and Barthold 2001). H. bilis mayalsocausemildhepatic firmed by PCR, immunohistochemistry, and other diagnostic lesions. studies. Some major infectious diseases are mentioned in the It is rare for H. hepaticus to cause liver lesions unless following to separate these background lesions from xenobio- the facility and animal room are known to be infected. If tic induced lesions. susceptible mouse strains are used in research, the more severe diffuse lesions may occur. Mouse infection in two- Helicobacter sp. Hepatitis (Figure 84; See Figure 87) year carcinogenesis bioassays has complicated interpreta- Pathogenesis: Infection by a number of different Helico- tion of carcinogenesis studies (Hailey et al. 1998; Stout bacter spp. et al. 2008).

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Murine Norovirus Hepatitis (See Figure 73) Comment: Mouse hepatitis virus (MHV), a coronavirus, infects hepatocytes, endothelium, and macrophages. In mouse liver, Pathogenesis: Infection by murine norovirus. virus strains may have different pathogenicities in the Diagnostic features: various mouse strains and in mice of different ages (Percy and Barthold 2007). Focal and multifocal necrosis is seen Focal, multifocal to diffuse areas of inflammation with multinucleated (syncytial) hepatocytes, endothelium, and sometimes with vasculitis in immunodeficient mice. macrophages. Immunodeficient mice may develop a chronic Focal to multifocal small areas of inflammation in persistent infection with chronic lesions in liver (Ward, Collins, most lines of mice. and Parker 1977). Inflammatory foci contain macrophages and Clinical MHV infection may be most commonly seen in lymphocytes. young mice. Adult mice may have serum antibodies but no Immunohistochemistry shows that inflammatory clinical signs and few, if any, histopathologic lesions. Some cells, especially macrophages in lesions and Kupffer cases show lesions in liver only. MHV is one of the most cells, can be positive for murine norovirus antigens. prevalent murine viruses in the United States and Europe (Homberger 1996) but appears less common today. Differential diagnosis: Tyzzer’s Disease (Clostridium piliforme Infection) Helicobacter hepatitis can appear similar but may Pathogenesis: Infection by Clostridium piliforme (Bacillus contain silver positive bacteria. piliforme). Helicobacter hepatitis is more common in A, BALB/ c, and C3H strains that are not susceptible to MNV Diagnostic features: infection. Inflammation, focal or multifocal, acute or chronic, Focal or multifocal necrosis, coagulation to caseous unknown etiology. with neutrophilic infiltration. Intrahepatocyte bundles of long basophilic bacilli. Comment: Murine norovirus (MNV) may cause severe hepa- Bacteria seen in Warthin-Starry, Giemsa stains, or by titis in some lines of immunodeficient mice but only minimal the PAS method. hepatitis or no lesions in most lines of infected immunocom- petent mice (Ward et al. 2006). MNV infection is the most Differential diagnosis: common viral infection in mouse colonies today. The impli- cations for interfering in experimental results are not known. Necrosis, of other known causes or unknown causes. It can be assumed that chemicals or infectious agents involving the immune system or liver may be influenced by MNV Comment: Named after Ernest Tyzzer who first described it infection. in a colony of Japanese walzing mice (Fox et al. 2002). Clos- tridium piliforme (Bacillus piliformis) is a long, thin, spore- forming (intracellular) bacterium. Rare lesion with sudden Mouse Hepatitis Virus Hepatitis (See Figures 63, 64, and 70) death, with or without diarrhea. Often infection of the colon with dissemination to the liver (focal hepatitis) and occasion- Pathogenesis: Infection by mouse hepatitis virus affecting ally heart (myocarditis). Special stains (e.g., Giemsa or hepatocytes, endothelium, and macrophages (Kupffer cells). Warthin-Starry Silver stain) can reveal intracytoplasmic fila- Diagnostic features: mentous bacteria. Tyzzer’s disease is rare in rodents. It is usually sporadic. Focal or multifocal hepatic necrosis. The gerbil is known to be extremely susceptible to infection Multinucleated cells (syncytia) from hepatocytes, (Fox et al. 2002). endothelium, and macrophages. Gross lesions can include hepatomegaly, focal necrosis, sin- MHV lesions in other tissues, including peritoneum, gle or multifocal, small or large lesions sometimes associated CNS, blood vessels. with lesions in other tissues, especially intestines (Percy and Chronic hepatitis and postnecrotic cirrhosis in immu- Barthold 2007). Multiple foci of necrosis (coagulative necro- nodeficient mice. sis) and/or multifocal necrotizing hepatitis can be observed microscopically. Differential diagnosis: E. Vascular Lesions Necrosis caused by toxins. Introduction Necrosis caused by murine norovirus. Necrosis caused by Helicobacter sp. The liver has a dual blood supply consisting of a relatively Necrosis caused by C. piliforme. major (about 75%) venous (portal) supply via the hepatic portal

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 24S THOOLEN ET AL. TOXICOLOGIC PATHOLOGY vein, which carries venous blood that is largely depleted of Angiectasis (Figures 86 and 87) oxygen, and relatively minor (about 25%) arterial blood sup- Synonyms: Peliosis hepatis, telangiectasis, sinusoidal dilation. ply, via the hepatic artery. The portal blood contains toxic materials absorbed in the intestine, and therefore the liver is the Pathogenesis: Perturbations in blood flow and/or drainage; first tissue to be exposed to toxic substances that have been weakening of sinusoidal walls. absorbed through the gastro-intestinal tract. Diagnostic features: Within the hepatic parenchyma, the hepatocytes are in intimate contact with the sinusoidal capillaries, which are car- Macroscopically—seen on the surface as blood- rying the mixture of blood originating from ramifications of filled, thin walled cavities projecting above the sur- the portal vein and hepatic artery to the central vein. The face (Bannasch et al. 1997). sinusoids are lined by modified endothelial cells containing Microscopically—There are two morphological fenestrations, which allow passage of lipoproteins and other types, as follows: large molecules but provide a barrier to blood cells. Kupffer 1. Cystic (‘‘Phlebectatic’’)—Focal dilation (disten- cells reside in the lumen of the sinusoids and are anchored sion) of endothelial lined channels (photographic to their wall. presentation contributed by Hardisty et al. 2007). The morphological aspect of the hepatic pathology during Can be an isolated lesion or have a multicentric circulatory disorders depends on the location of the vascular form. The lacunae are densely packed with blood structure being affected (i.e., lobular sinusoids, the outflow and are coated by a single layer of endothelium hepatic vein, or the inflow portal vein). and separated from one another by cords of liver parenchyma (Bannasch, Wayss, and Zerban 1997). The endothelial cells appear to be unal- Congestion (Figure 85) tered, and there is no increase of mitotic figures. Synonym: Chronic passive congestion. The tissue adjacent to the dilated sinusoids is well preserved and free of necrotic cells. Pathogenesis: Circulatory failure, typically right-sided heart 2. Cavernous (‘‘Parenchymal’’)—The peliotic cysts failure. are not, or are only partially, lined by endothelium. Thus, the cysts involve not only the sinusoidal Diagnostic features: lumen, but also the space of Disse, and the blood comes in direct contact with the neighboring par- Increase prominence (number) of erythrocytes in the enchymal cells. The parenchyma undergoes focal capillary bed or larger vessels of an organ. necrosis without a zonal distribution. This lesion is No appreciable distention (angiectasis) of the vessel much less likely to be preneoplastic and a few tox- wall. ins have been shown to induce the lesion. When Diagnosis often correlates with gross observation endothelial lining is absent, the term Peliosis (e.g., reddened, darkened focus). Hepatis instead of Angiectasis is indicated. May be associated with centrilobular necrosis. Differential diagnosis: Differential diagnosis: Hemangioma—expansile structure lined by flattened Angiectasis—dilated vascular spaces lined by endothelial cells; may be associated with parenchy- endothelial cells; dilated vascular channels and mal compression. spaces frequently contain erythrocytes. Cystic degeneration (Spongiosis hepatis)—cavities Massive necrosis. are not filled with blood but with a finely flocculent Hemorrhage-irregular patchy lakes of blood not con- acidophilic material. tained within defined vascular channels. Autolysis-altered cellular texture and loss of staining Comment: Angiectasis is a cystic or cavernous widening of the intensity. liver sinusoids that can occur in a variety of pathological insults. In human, sinusoidal dilatation has been reported fol- Comment: Congestion may result from circulatory disturbance lowing hypoxia or hyperperfusion as a result of right-sided such as right-sided heart failure and is usually associated with heart failure, thrombosis of hepatic veins, amyloid deposition, necrosis of the centrilobular areas (Burt, Portmann, and granulomatous, or neoplastic disease (Greaves and Faccini MacSween 2002). Presence of blood in hepatic sinusoids seen 1992; Bruguera et al. 1978). Although these lesions can also in animals that die or in situations where there is incomplete occur spontaneously in different rodents with different diseases exsanguination should not be diagnosed as congestion. If it is or with certain neoplasms causing hemodynamic changes, it absolutely necessary to record such findings, it is important can also be induced by different compounds. Focal sinusoidal to qualify the blood stasis in the liver as passive congestion. dilatation and peliosis hepatis have been observed in the rodent

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 25S liver after treatment with nitrosamines, pyrrolizidine alkaloids Comment: There are several potential mechanisms leading to or glucocorticoids (Greaves 2007; Ruebner, Watanabe, and liver thrombosis. Activation of the coagulation system associ- Wand 1970; Ungar 1986; Wolstenholme and Gardner 1950). ated with fibrin deposits and hypoxia located in the centrilob- Altered hemodynamic and changes in the hepatic microcircula- ular sinusoids was reported to occur in the livers of rats tion have been proposed to be of importance in the pathogen- exposed to monocrotaline (MCT) (Copple et al. 2002). It was esis of sinusoidal dilatation (Slehria et al. 2002). Subcapsular suggested that the fibrin thrombi were formed following sinusoidal dilatation can also be found a postmortem finding chemical-induced hepatic endothelial cell damage. In studying in rats (Kimura and Abe 1994). an endotoxin-exposure model, it was suggested that the noted Angiectasis is defined by multiple blood-filled cystic spaces focal and random hepatocellular necrosis was caused by circu- of different size and shape occurs after suspected loss or weaken- latory disturbances due to fibrin thrombi in clusters of adjacent ing of sinusoidal walls and/or supporting tissue. The two subtypes sinusoids. Using a rat model of 2-butoxyethanol induced hemo- may occur in combination. The cystic spaces are devoid of lytic anemia associated with systemic thrombosis, fibrin endothelial lining in the cavernous subtype (‘‘parenchymal’’) thrombi were noted in the central vein and sinusoids of the although controversy still exists (Greaves 2007; Edwards, liver, in addition to the presence of thrombi seen in several Colombo, and Greaves 2002). Angiectasis can be found in aged other organs (Ramot et al. 2007). rats (Lee 1983). However, these lesions can also be induced in both rats and mice after viral infection (Bergs and Scotti 1967) Infarction (Figures 89 and 90) or exposure to certain drugs or chemicals (Mendenhall and Pathogenesis: Interruption of blood flow in a major vessel. Chedid 1980; Husztik, Lazar, and Szabo 1984). Torsion of hepatic lobe. Angiectasis has also been reported in animals or humans Diagnostic features: infected with Bartonella spp. (Wong et al. 2001; Breitschwerdt and Kordick 2000) and it has been associated with a number of Extensive area of necrosis may be associated with diseases in humans as well as administration of anabolic steroids inflammation. and oral contraceptives (Naeim, Cooper, and Semion 1973; The necrosis does not have acinar pattern. Zimmerman 1998; Tsokos and Erbersdobler 2005). Angiectasis may refer to a vascular lesion formed by dilatation Differential diagnosis: of a group of small blood vessels. It can be observed in transgenic mouse models (Srinivasan et al. 2003; Bourdeau et al. 2001) or Hepatocellular necrosis resulting from direct toxicity related to xenobiotic administration in rats and mice (Robison may have a diffuse or lobular distribution but also may et al. 1984; Kim et al. 2004). It can be found as an incidental find- be accompanied by infarction; not mutually exclusive. ing in aging mice and is sometimes associated with hepatocellular neoplasms (Harada et al. 1996, 1999). Angiectasis can be Comment: Aside from torsion of liver lobes, which can occur chemically induced (Bannasch, Wayss, and Zerban 1997) and spontaneously in rodents, infarction is a very rare lesion that has been suggested to be preneoplastic in some animal models. can be induced only under very specific experimental conditions. The combined injection in mice of NG-monomethyl-L- Thrombosis (Figure 88) arginine and aspirin after lipopolysaccharide exposure resulted in significant hepatocellular enzyme release, characterized his- Pathogenesis: Activation of the coagulation system associated tologically by intravascular thrombosis with diffuse infarction with arteritis or phlebitis or secondary to atrial thrombosis. and necrosis (Harbrecht et al. 1994). Intraperitoneal injections Diagnostic features: in rats of vasoconstrictor xenobiotics such as phenylephrine produced infarcts of the spleen regularly and infarcts of the It is characterized by the formation of a thrombus liver occasionally (Levine and Sowinski 1985). Isolated perfu- within the lumen of a blood vessel, like sinusoids and sion with 1.0 g/kg of the cytotoxic xenobiotic 5-FU or central veins. hyperthermia of 41 degrees C 10 min resulted in 90% to Amorphous mass attached to the endothelium or free 100% mortality in rats, with extensive, patchy necrosis, and within the blood vessel lumen (due to plane of section). infarction on histologic examination (Miyazaki et al. 1983). Contains fibrin, platelets, and entrapped blood cells. 3 Damaged endothelium can be seen. Endothelial Cell Hypertrophy/Karyomegaly (Figure 91) May occur with histiocytic sarcoma in rats or mice. Synonyms: Endothelial cell enlargement, cytomegaly.

Differential diagnosis: Introduction: This is a relatively new diagnostic entity. Because of the difficulty of identifying specific sinusoidal cell Postmortem clot. Necrotic area of tissue—loss of microscopic tissue 3 The examples provided were confirmed with special stains (not shown). structure and stain affinity; pale eosinophilic stain- However, based solely on H&E staining, a diagnosis of ‘‘sinusoidal cell ing, and absence of nuclear detail. hypertrophy/karyomegaly’’ is appropriate.

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Pathogenesis: Continued DNA synthesis and cell cycle arrest Pathogenesis: Proliferation of normally present sinusoidal lin- following exposure to certain xenobiotics. ing endothelial cells without sinusoidal dilation. Diagnostic features: Diagnostic features:

Irregularly shaped karyomegalic nuclei. Focal, located to the portal triad, consisting of Nuclei and cells appear larger than normal. increased number of capillaries. Secondary changes related to the endothelial Blood may not be present within the capillaries. cell damage and/or obstruction of the sinusoids lead- Absence of supporting tissue. ing to local ischemia may be seen. Such changes May have mitotic figures and nuclear atypia. include sinusoidal congestion, hemorrhage, thrombi formation, hepatocytic fatty change/degeneration, Differential diagnosis: and necrosis (Lailach et al. 1977; Nyska et al. 2002). Hemangioma—expansile structure lined by flattened Differential diagnosis: endothelial cells; may be associated with parenchymal compression. Endothelial cell hyperplasia characterized by Angiectasis—dilated vascular spaces lined by increased number of endothelial cells. endothelial cells; dilated vascular channels and Hepatocytic cytomegaly and karyomegaly. spaces frequently contain erythrocytes. Anisokaryosis—variation in the size of nuclei and/or binuclearity admixed with diploid appearing hepato- Comment: Endothelial derivation and proliferation can be cyte nuclei. confirmed by dual immunostaining for CD31/KI-67 (Ohnishi Reactive Kupffer cells—enlarged histiocytic cells et al. 2007). Comparative endothelial cell kinetic studies in with visible cytoplasm lining sinusoids. human, mice, and rats indicated that the labeling index (LI) Histiocytic sarcoma—hepatic parenchyma infiltrated in the male and female B6C3F1 mouse liver was significantly and replaced by collections of histiocytic cells that higher (p <.01)comparedtotheLIinmaleandfemalerat can be pleomorphic. and human liver, and the LI in the male and female rat liver was significantly greater (p < .05) than the LI in human liver. It was suggested that the increased rate of spontaneous Comment: In a study of monocrotaline (Wilson et al. 2000) it hemangiosarcoma formation in mice may be related to the was suggested that the endothelial karyomegaly was the result increased proliferation rate of endothelial cells normally of continued DNA synthesis and concentration-dependent cell- present in the B6C3F1 strain of mice compared to rats and cycle arrest. The exposed cells undergo a process of multiplica- humans (Ohnishi et al. 2007). tion of chromosomal copies, defined as endopolyploidy, with nuclear and cytoplasmic gigantism. A direct correlation between cytoplasmic volume and nuclear DNA content was F. Non-Neoplastic Proliferative Lesions suggested. Introduction A similar pathogenesis was suggested in the case of ridelliinne-induced endothelial cytomegaly and karyomegaly A variety of non-neoplastic proliferative lesions of different (Nyska et al. 2002). Administration of riddelliine, a naturally origin(s) occurs spontaneously in the liver of rodents and may occurring pyrrolizidine alkaloid, to rats results in cytomegaly and also be induced by treatment with chemicals. Incidences and karyomegaly of hepatic endothelial cells as one of its pleotrophic morphological characteristics vary considerably by animal spe- responses to cell-specific cytotoxicity. A metabolite of this cies, strain, and sex. Some of these lesions might be regarded as pyrrolizidine alkaloid is believed to directly interact with pre-neoplastic alterations. endothelial cell DNA. As a non-neoplastic proliferative response, increased Sometimes this change is interpreted as prominent hepatocyte mitoses (Figure 92) above normal background Kupffer cells or Kupffer cell hypertrophy on H&E sections levels or increased above what is seen in control animals can and warrants further characterization for confirmation of occur in rodent livers. Causes vary from physiological the endothelial cell origin. Immunohistochemical stains responsessuchasduringearlygrowth, during pregnancy, and andelectronmicroscopycanbeused to identify the cell type following partial hepatectomy to post-necrotic repair. This involved. change may be diagnosed as cytologic alteration or mitotic

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 27S alteration with a description in the pathology narrative. Portal areas and central veins not present in small foci In some laboratories diagnosis of ‘‘increased mitoses’’ is but can be seen in larger foci. used. Sinusoids within focus may be compressed, so typical parenchymal plates are difficult to detect. Tortuous hepatic cords may occur due to increased Focus of Cellular Alteration number of cells. Size of cells and cytoplasmic tinctorial variation Introduction: Foci of cellular alteration are common in rodent depend on type of focus. studies greater than duration of twelve months and may be Normally absence of cellular atypia. seen in short duration toxicity studies following exposure Cystic degeneration (spongiosis hepatis) and to certain xenobiotics. Foci of cellular alteration can be iden- angiectasis (peliosis hepatis) may occur within foci tified by special stains. In H&E-stained slides they may be of altered cells. subclassified based on the predominant cell type present. Cytoplasmic lipid may be present. Diagnosis of the mixed cell subtype of altered hepatic focus Intracytoplasmic inclusions of various types may be varies among different laboratories. One viewpoint defines a present. mixed focus of cellular alteration as consisting of a combination of basophilic, vacuolated, eosinophilic, and/or clear cell hepatocytes without a predominant cell type. An alternative viewpoint defines a mixed cell focus as containing any two Basophilic (Figures 93–97) phenotypes of cells in approximately a 50%/50% proportion. Basophilic, diffuse (Figures 93 and 94). Others regard a ‘‘true’’ mixed focus as one that contains clearly identified basophilic and eosinophilic cells regardless Hepatocytes of normal size or slightly enlarged with of the proportions of each. Because of this diverse set of homogeneously staining cytoplasmic basophilia due diagnostic opinions regarding focus subtypes, the pathologist to abundant free ribosomes. is encouraged to describe the morphological features Cells may be pleomorphic with enlarged vesiculated of documented foci in detail, especially if they are altered nuclei and prominent nucleoli. by treatment. Dissociation of cells may occur. Mitotic figures may occur. Synonyms: Areas of cellular alteration; focus of altered Basophilic, tigroid (Figure 95). hepatocytes; hyperplastic focus; preneoplastic focus; enzyme altered focus; phenotypically altered focus. Cells are usually smaller; enlarged cells may occur. Pathogenesis: A localized proliferation of hepatocytes pheno- Cells are often arranged as tortuous cords. typically different from surrounding hepatocyte parenchyma. Cells display large abundant basophilic bodies often arranged in clumps or long bands with striped pattern Diagnostic features: in paranuclear or peripheral regions of cytoplasm (due to increased rough endoplasmic reticulum). May occasionally be observed grossly as small white Mitotic rate may be increased. foci on the liver surface, but not round nodules. Size may range from less than one lobule to multiple Basophilic, NOS (Figure 96). lobules in diameter. Foci that are not clearly tigroid or diffusely Circular or ovoid shape; irregular formed foci may basophilic. occur. Peliosis or spongiosis may occur within these foci. Distinguished into types of foci by virtue of tinctorial variation, size of hepatocytes, and textural appear- Basophilic (no further classification in mice) (Figure 97). ances from surrounding parenchyma. May be subclassified based on predominant cell type. Consist of cells larger or smaller than normal hepato- The fact that 80% of the focus is composed of one cytes, in general they are smaller. morphologic cell type (basophilic, eosinophilic, etc.) Cytoplasm exhibits distinct basophilia due to free or a mixed cell type. ribosomes or rough endoplasic reticulum. Normally no or only minimal compression of the sur- Often cells contain obvious glycogen. rounding liver tissue. Intracytoplasmic basophilic clumps with relatively Liver plates merge imperceptible with surrounding clear intervening cytoplasm or the cytoplasmic baso- hepatic parenchyma; nevertheless foci are sharply philia may be distributed homogeneously. demarcated from the adjacent normal hepatocytes Vascular pseudo-invasion may be present. by the appearance and staining reaction of its cells. Eosinophilic cytoplasmic inclusions may be found Lobular architecture preserved. occasionally within hepatocytes.

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Eosinophilic (Figures 98–100) the surrounding parenchyma. Distinct compression of adjacent normal hepatocytes is present. Synonyms: Acidophilic, ground glass. Carcinoma, hepatocellular—distinct trabecular, adenoid or poorly differentiated growth is present. Composed of usually enlarged, polygonal hepato- Lobular and plate architecture is not maintained. cytes with acidophilic staining cytoplasm. Cellular atypia and invasive growth may be present. Some clear cells may be present. Cytoplasm is distinctly granular and pale pink, inten- Comments: Foci of cellular alteration represent a localized pro- sely eosinophilic, or shows ground-glass appearance. liferation of hepatocytes that are phenotypically different from Nuclei often enlarged; nucleoli may be prominent surrounding hepatocyte parenchyma. They are subclassified and centrally located. based upon phenotypic and tinctorial features. Foci of cellular Eosinophilia may result from an increase in smooth alteration can occur spontaneously in aged rats and other endoplasmic reticulum, peroxisome, or mitochondria rodents and can be induced by chemical treatment. The inci- (rats, mice). dence, size, and/or multiplicity of foci are usually increased Store glycogen in excess. and time to development usually decreased after administration of hepatocarcinogens (Hanigan, Winkler, and Drinkwater Mixed Cell (Figures 101–104) 1993; Frith, Ward, and Turusov 1994; Bannasch and Zerban Synonyms: Basophilic/eosinophilic mixed. 1990; Moore, Thamavit, and Bannasch 1996). Foci of cellular Heterogeneous focus consisting of a combination of cell alteration are not necessarily preneoplastic. Foci of cellular types (see Introduction above). alteration may have prominent fat deposition and characteristic features of cystic degeneration and angiectasis (See B-focus Clear Cell (Figures 105–107) with spongiosis; Figure 96). Foci can be subclassified based on the predominant cell Composed of normal-sized or enlarged cells with dis- type. If no single cell type comprises at least 80% of a given tinct cytoplasmic clear spaces. focus, it should be classified as mixed. Mostly these mixed foci Some eosinophilic or basophilic cells may be present. consist of both basophilic and eosinophilic/clear type cells. In Nuclei are often small and dense, prominent and these foci, it is not clear what is the predominant phenotype and centrally located, sometimes exhibiting increased are therefore indicated as ‘‘mixed.’’ volume. Species and strain differences occur in the prevalence of Store glycogen in excess. these foci. It is not uncommon for a focus predominantly Cell membranes may appear prominent. of one cell type, however, to have a small number of a different type. Amphophilic (in Rats and Mice) (Figure 108) Mixture of eosinophilic and clear cells can be classified into Hepatocyte cytoplasm having an affinity both for either eosinophilic or clear cell focus in accordance with pro- acid and for basic dyes. portion of clear cells. Mixture of amphophilic and other pheno- Large cells with homogeneous, granular intensely types has never been observed in rodents. eosinophilic cytoplasm, and randomly scattered faint Usually, amphophilic foci are less frequently observed in basophilia. rats and mice compared to other foci. Nuclei slightly enlarged. A number of models have linked specific types of foci of Poor in or free of glycogen. cellular alteration with carcinogenesis (Mahon 1989). The Amphophilic (basophilic and eosinophilic) cytoplasm nitrosomorpholine model is linked with eosinophilic and clear due to a proliferation of both mitochondria and rough cell foci as precursors. Aflatoxin is linked with basophilic foci endoplasmic reticulum (mitochondrial-RER complex). as a tumor precursor (Bannasch, Zerban, and Hacker 1985). Usually less frequently observed in rats and mice It was also reported that hepatocarcinogenesis was associated compared to other foci. with increase of basophilic or amphophilic foci (Goodman et al. 1994). Although age-related eosinophilic or tigroid Differential diagnosis: basophilic foci were not associated with exposure to hepatocar- Hyperplasia, hepatocellular, regenerative—evidence cinogens, in hamsters, treatment with nitrosamines or other of prior or ongoing hepatocellular damage. carcinogens caused a variety of foci including basophilic foci Hyperplasia, hepatocellular, non-regenerative— (Frith, Ward, and Turusov 1994; Moore, Thamavit, and usually seen as a single large nodule of hyperplasia Bannasch 1996). As some foci may be potential precursors of without concomitant evidence of hepatocellular dam- neoplastic formation, careful identification of altered foci is war- age or phenotypical alteration. Rare in rats and mice. ranted (Maronpot et al. 1989). Although induced by carcinogens, Adenoma, hepatocellular—loss of normal lobular foci of cellular alteration can be found as non-neoplastic end architecture with irregular growth pattern. Liver stage lesions and not all foci can be related to carcinogens (Per- plates often impinge perpendicular or obliquely on aino et al. 1984; Harada, Maronpot, Morris, Stitzel, et al. 1989;

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Harada, Maronpot, Morris, and Boorman, 1989; Squire 1989; treatment-associated and consists of a proliferative collection Schulte-Hermann et al. 1989). Most importantly, types of of hepatocytes spanning several lobules and without evidence foci in controls should be compared to those found in treated ani- of prior hepatic damage. This lesion is not associated with any mals. Some pathologists regard vacuolated foci as focal fatty evidence of existing or prior hepatocellular injury. Diagnostic change and do not consider them a subtype of focus of cellular difficulties occur when preneoplastic foci and hepatocellular alteration. adenomas occur in the same liver sections of older rodents. This lesion may be similar to that of hepatic nodular hyperpla- Hyperplasia, Hepatocellular, Non-Regenerative (Figures sia in dogs. 109 and 110) There are basically two variations of non-regenerative hepatocellular hyperplasia. One is relatively smaller and is Synonyms: Hyperplasia, hepatocellular, focal hepatocellular accompanied by angiectasis and/or spongiosis hepatis and the hyperplasia. other tends to be larger than several lobules. The former occurs in both sexes and the latter predominantly in untreated Pathogenesis: A spontaneous or treatment-associated prolif- female control F344 rats but occasionally reported in treated erative collection of hepatocytes spanning several lobules and rats(Tasakietal.2008;Haileyetal.2005;Bachetal. without evidence of prior hepatic damage. 2010). When present near the capsular surface, this type of Diagnostic features: nodular hyperplasia may be evident grossly as a raised area. The proliferating cell nuclear antigen (PCNA) labeling index A relatively large lesion that is often greater than sev- in these nodules is increased in comparison with surrounding eral adjacent lobules and is occasionally accompa- parenchyma and the lesion is glutathione S-transferase pla- nied by angiectasis and/or spongiosis hepatis (cystic cental form (GSTP) immunonegative. degeneration). Very early non-regenerative hyperplasia may be the size of Comprised of slightly enlarged hepatocytes. small foci of cellular alteration and are identified by their Hepatocytes are tinctorially similar to surrounding altered growth pattern and tinctorial similarity to surrounding parenchyma. parenchyma (see Figure 109). The liver plates in the lesion tend to merge with the adjacent hepatic parenchyma. May be minimal to mild compression of adjacent Hyperplasia, Hepatocellular, Regenerative (Figures 111–113) hepatic parenchyma. Synonyms: Hyperplasia, hepatocellular; hyperplasia, regenera- Lobular architecture is maintained. tive; hyperplasia, nodular; regeneration, nodular. Portal triads and central veins are present. When accompanied by angiectasis/spongiosis hepa- Pathogenesis: A nodular regenerative response to prior or con- tis, hepatic cords may be distorted. tinuous hepatocellular damage. Differential diagnosis: Diagnostic features:

Focus of cellular alteration—phenotypical or tinctor- Focal or multifocal (nodular) appearance. ial variation is present. Generally not associated with Lesion may reach several millimeters in diameter. chronic liver damage, though foci of cellular Spherical proliferation may be accompanied by slight alteration may occur in damaged liver. encapsulation. Adenoma, hepatocellular—loss of normal lobular Compression of surrounding liver parenchyma often architecture with irregular growth pattern. Liver plates occurs. often impinge perpendicular or obliquely on the sur- Normal lobular architecture usually present but may rounding parenchyma. Distinct compression is present. be distorted. Carcinoma, hepatocellular—distinct trabecular, ade- Portal triads and central veins may be present. noid, or poorly differentiated growth is present. Bile duct and oval cell proliferation may be Lobular and plate architecture is not maintained. present. Cellular atypia and invasive growth may be present. Hepatocytes appear slightly altered, but may have Hyperplasia, hepatocellular (regenerative)—evidence slightly basophilic cytoplasm or prominent nucleoli. of ongoing or prior hepatocellular damage, fibrosis, Increased mitotic index may be observed. or a history of exposure to a known hepatotoxicant. Evidence of prior or ongoing hepatocellular damage, Lobular architecture usually distorted. such as apoptosis/necrosis, chronic inflammation, chronic congestion, fibrosis, cirrhosis, or a known Comment: Non-regenerative hepatocellular hyperplasia cause of toxicity. is rare in rodents. It may occur spontaneously or be Lesions in rats tend to be more nodular than in mice.

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Differential diagnosis: Differential diagnosis:

Focus of cellular alteration—tinctorial variation is Infiltration, mononuclear—well demarcated; usually present. Generally not associated with chronic liver solitary; associated with cell necrosis. damage, though foci of cellular alteration may occur Histiocytic sarcoma—diffuse and irregular prolifera- in damaged liver. tion of histiocytic cells throughout the liver. Usually Adenoma, hepatocellular—loss of normal lobular associated with destruction of hepatic parenchyma. architecture with irregular growth pattern. Liver Occasional multinucleated giant cells may be plates often impinge perpendicular or obliquely on present. the surrounding parenchyma. Distinct compression Hyperplasia, oval cell—consists of a single or double is present. row of oval cells sometimes forming incomplete Carcinoma, hepatocellular—distinct trabecular, (pseudo-) duct-like structure. Cells are usually uni- adenoid, or poorly differentiated growth is present. form in size and shape and have scant pale basophilic Lobular and plate architecture is not maintained. Cel- cytoplasm and round to oval nuclei. lular atypia and invasive growth may be present. Hyperplasia, hepatocellular (non-regenerative)—no Comment: Rare as a spontaneous finding, hyperplasia of history or evidence of hepatocellular damage. Kupffer cells may be seen following phagocytosis of foreign material and as a consequence of estrogen treatment and in Comment: These lesions are considered to represent a regen- inflammatory conditions. It can be induced by cytokines. erative response to prior or continuous hepatocellular dam- Hypertrophy and hyperplasia often accompany each other. The age. A history of exposure to a hepatotoxicant and the hypertrophy of normal Kupffer cells gives the impression of presence of multiple nodules of regeneration that maintain a presence of more Kupffer cells since they are difficult to visua- lobular but usually distorted architecture makes diagnosis lize in normal livers. more convincing. Diagnostic difficulties occur when preneoplastic foci and hepatocellular adenomas occur in the same liver sections of older rodents or in livers with many induced Hyperplasia, Ito Cell (Figures 116–118) foci and tumors. Synonyms: Stellate cell; perisinusoidal cell; fat-storing perisi- In livers with partial hepatectomy (PH), the pattern of nusoidal cell. hyperplasia at twenty-four to seventy-two hours post surgery is diffuse hepatocyte hyperplasia with many mitotic figures and Pathogenesis: Proliferation of fat-storing perisinusoidal cells. no evidence of liver toxicity. Although this response is also hyperplasia (hepatocellular, regenerative), it is not to be con- Diagnostic features: fused with the nodular hepatic response to toxic damage to hepatocytes. After ninety-six hours, the liver may be almost Focal or diffuse proliferation of Ito cells. normal histologically. In mice however, chronic biliary lesions May grow in sheets, clusters, or along cords of may be seen in the liver after PH. hepatocytes. Cells vary in size and shape and are vacuolated. Multiple cytoplasmic fat droplets of different size Hypertrophy/Hyperplasia, Kupffer Cell (Figures 114 and 115) occur. The nuclei are ovoid or round and may be indented by Synonyms: Kupffer cell proliferation; histiocytosis, focal or cytoplasmic lipid droplets. diffuse. Modest amount of collagenous matrix may be present. Pathogenesis: Following phagocytosis of foreign material, Differential diagnosis: estrogen treatment, inflammatory conditions, and response to cytokines. A rare spontaneous finding. Fatty change/Lipidosis—the cytoplasm of fat cells Diagnostic features: may be clearer than that of Ito cells. Ito cell tumor—larger and more extensive than Diffuse to multifocal proliferation of oval to spinde- hyperplasia. Partially distinct compression of adja- loid cells lining sinusoids. cent hepatic parenchyma. Cells resemble histiocytes and often contain phagocytic material. Comment: Ito cell hyperplasia is extremely rare and occurs May form as sheets or nodules. predominantly in mice. It arises from fat-storing perisinusoi- Hypertrophy can occur without hyperplasia and vice dal cells, better known as Ito cells (Dixon et al. 1994; Enzan versa. 1985; Tillmann et al. 1997). The biological behavior of the

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 Vol. 38, No. 7S, 2010 LESIONS OF THE HEPATOBILIARY SYSTEM 31S lesion is not well established. There appears to be a continuum Diagnostic features: with Ito cell tumor (see the following), which may be just an exaggerated and sometimes more localized form of Ito cell Consists of dilated to cystic bile ducts filled hyperplasia. with mucus and cellular debris and surrounded by inflammatory cell infiltrates and connective Bile Duct Hyperplasia (Figures 119–122) tissue. Glandular epithelium is typically a single layer and Pathogenesis: A spontaneous change in portal areas of older varies from flattened to tall columnar hyperbasophi- animals; may be induced or exacerbated by treatment. lic and pleomorphic cells along with goblet cells and Diagnostic feature: occasional Paneth cells. Glandular epithelium, particularly in cystic glands, Increased number of small bile ducts arising in portal may be partially lost through degeneration resulting region. in crescent shaped structures. May not involve all portal areas. Central portions of large lesions may become sclero- May be associated with periductular fibrosis and tic with only remnants of biliary epithelium suggest- periductular cell infiltration. ing regression. Biliary epithelium is well differentiated, forming Lesions may be limited to small foci but may occupy normal ducts. large interconnecting areas of a lobe without mark- Biliary epithelium may show degenerative or edly disturbing the lobe outline. atrophic changes. Lesion growth typically involves contraction with May be associated with oval cell hyperplasia. retraction of surrounding parenchyma. Markedly May contain mucous metaplasia or hyalinosis in dilated or cystic mucus-filled glands along the liver specific situations in mice. capsule may protrude above the lobe outline. Cystic form. Older lesions may be shrunken from the liver surface Focal bile duct proliferation with cystic dilation of and appear as scars. ducts may occur. Regenerative hepatocellular hyperplasia may Usually acini lined by flattened epithelium. be present when there is extensive parenchymal involvement. Differential diagnosis: Differential diagnosis: Cholangioma—well demarcated; usually solitary; acini lined by cuboidal epithelium. Cholangiocarcinoma—solid sheets, trabeculae, or Cholangiofibrosis—central portions become nests of closely packed biliary cells largely displacing atrophic, collagenized, and avascular whereas the hepatic parenchyma. Intestinal metaplasia is not a active proliferating portion is at the periphery; mucus prominent feature. Glandular dilation is absent or production is common. minimal. Cholangiocarcinoma—invasive growth into sur- Hyperplasia, bile duct—multiple small bile ducts rounding hepatic tissue or vessels; cell are pleo- arising in portal region. Biliary epithelium is well morphic; mucus production. differentiated, forming more normal appearing Oval cell hyperplasia—consists of a single or double ducts, with minimal glandular dilation in some row of oval cells forming incomplete duct-like struc- cases. tures. Cells usually uniform with scant basophilic cytoplasm and round or oval nuclei. Comment: This lesion is an inflammatory, proliferative, and metaplastic reaction involving bile duct epithelium and is Comment: Often associated with evidence of hepatic injury and seen with hepatocellular toxicity caused by xenobiotics such repair and obstruction of bile flow. Dilation of intrahepatic bile as dioxins, furans, and related chemicals in rats (Bannasch ducts is a spontaneous, age-associated lesion that is more com- and Zerban 1990; Deschl et al. 1997; Eustis et al. 1990; mon in rats than in mice. Kimbrough et al. 1973; Kimbrough and Linder 1974; Sirica 1992; Hailey et al. 2005). The initial reaction following pronounced hepatic parenchymal necrosis is oval cell hyperplasia Cholangiofibrosis (Figures 123–126) (Engelhardt 1997). Synonyms: Bile duct adenomatosis; intestinal cell metaplasia; Cholangiofibrosis is a controversial lesion that has been adenofibrosis. diagnosed as cholangiocarcinoma especially when there is extensive involvement of the liver. Cholangiofibrosis is not Pathogenesis: Originates from an initial oval cell hyperplasia seen as a spontaneous lesion but occurs primarily in rats treated in response to pronounced hepatic parenchymal necrosis. with a variety of xenobiotic agents that are hepatotoxic at high

Downloaded from tpx.sagepub.com at Society of Toxicologic Pathology on May 21, 2015 32S THOOLEN ET AL. TOXICOLOGIC PATHOLOGY dose. The intestinal cell metaplasia in cholangiofibrosis hepatocellular neoplasms and may play an important role in includes goblet and Paneth cells and columnar cells similar hepatocarcinogenesis. Some authors support the concept that to chief cells identified in H&E-stained paraffin sections and oval cells may participate in the lineage of hepatocellular and enterochromaffin cells identified by special staining. The cholangiocellular carcinomas and may serve as hepatic stem metaplastic cells have been confirmed by electron micro- cells. Oval cell hyperplasia diagnosis including grade is rec- scopy in some cases. While cholangiofibrosis has been ommended, even when it is part of a complex set of hepatic reported to progress to cholangiocarcinoma with malignant changes. features (Bannasch and Zerban, 1990; Sirica 1992), unequivocal metastases have not been confirmed in most cases. This G. Neoplasms lesion is not observed in humans. Introduction Oval Cell Hyperplasia (Figures 127–129) The rodent liver is the most common target site of chemical Synonyms: Oval cell proliferation; bile ductule cell hyperplasia. carcinogens (Maronpot et al. 1986; Evans and Lake 1998), per- haps due to its major function as a metabolizing and detoxifying Pathogenesis: Arises from terminal ductule epithelial cells organ for xenobiotics. Rodent hepatocarcinogens are usually (canal of Hering cells) spontaneously, following liver infec- hepatotoxins. The chronic toxicity of these toxins may tions, and secondary to hepatotoxic injury. contribute to hepatocarcinogenesis although genotoxic liver Diagnostic features: carcinogens are often also hepatotoxins. There is over a thirty- year history of experimental induction (Frith and Wiley 1982; Generally originates from portal areas and is often Malarkey et al. 1995; Evans et al. 1992; Ward et al. 1983, multifocal. 1986; Ward, Lynch, and Riggs 1988; Popp 1984) and classifica- Consists of a single or double row of oval to round tion of preneoplastic and neoplastic lesions of the rat and mouse cells along sinusoids in linear arrays. liver in book chapters (Bannasch and Zerban 1990; Brooks May form a few or many small ductules with stream- and Roe 1985; Greaves and Faccini 1984; Jones and Butler ing into the hepatic parenchyma. 1978; Ward 1981; Harada et al. 1999; Eustis et al. 1990) and Formation of incomplete duct-like structures may by committee (ILAR 1980) or toxicologic pathology be present. societies (Standardized System of Nomenclature and Diagnos- Cells are usually uniform in size and shape and may tic Criteria [SSNDC] Guides, http://www.toxpath.org/ be fusiform. ssndc.asp). Terminology has evolved to the present nomencla- Cells have scant pale basophilic cytoplasm and round ture that is also based on many publications on liver or oval nuclei. carcinogenesis. Oval cells express keratin. There is evidence from experimental studies documenting the regression of proliferative hepatocellular lesions including Differential diagnosis: foci of cellular alteration, hepatocellular adenomas, and hepatocellular carcinomas following cessation of treatment (Maronpot Hyperplasia, bile duct—several small bile ducts 2009). A dramatic example was reported in mice following arising in portal region. Biliary epithelium is well cessation of chronic chlordane exposure (Malarkey et al. differentiated, forming normal ducts. 1995). Similar experience has been reported in rats and mice Early or mild fibrosis—collagen matrix is evident. in other studies (Lipsky et al. 1984; Greaves, Irisarri, and Inflammation—presence of mononuclear, polymor- Monro 1986; Marsman and Popp 1994) as well as in humans phonuclear, or connective tissue cells will be present (Fre´mond et al. 1987; McCaughan, Bilous, and Gallagher in inflammation. 1985; Emerson et al. 1980; Steinbrecher et al. 1981). Agents that require continual administration for the stable presence and Comment: Oval cell proliferation is considered to arise from growth of preneoplastic and neoplastic rodent liver lesions can terminal ductule epithelial cells (canal of Hering cells). It is be categorized as conditional hepatocarcinogens (Maronpot a rare spontaneous lesion in rats. Oval cell hyperplasia can 2009). be observed following severe hepatotoxic injury and treatment with hepatocarcinogens. There is often a close relation Hepatocellular Adenoma (Figures 130-134) to the portal tract, although more scattered groups of proliferating oval cells can be seen diffusely throughout the liver Synonyms: Adenoma, hepatic; adenoma, liver parenchymal following xenobiotic-induced hepatic injury (Engelhardt cell; hepatoma, benign; tumor, liver cell, benign; hepatoma, 1997). benign; type A nodule. In mice oval cell hyperplasia is a feature of chronic active hepatitis caused by H. hepaticus and H. bilis and is seen fol- Pathogenesis: Spontaneous and following treatment with lowing treatment with various hepatocarcinogens. Hyperplas- hepatotoxins that are carcinogenic xenobiotics; with gene tic oval cells occur in association with a high incidence of alterations in genetically engineered mice.

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Diagnostic features: and/or occurrence of cellular atypia; adenoid, trabecular, or poorly differentiated growth. Are often but not always grossly visible as small (1 mm) to large uniformly round nodular lesions. Comment: Hepatocellular adenomas occur spontaneously with Histologically lesions are nodular and compressing increased incidence in older rodents and following treatment of adjacent normal hepatocytes. with hepatotoxins that are carcinogenic xenobiotics (Harada Sharply demarcated from surrounding liver et al. 1999; Eustis et al. 1990; Stinson, Hoover, and Ward parenchyma that is often compressed. The compres- 1981). In mice, hepatocellular carcinomas can be seen histologi- sion of adjacent normal hepatocytes should be at least cally arising within adenomas of both spontaneous and induced on two quadrants of the adenoma. adenomas (Jang et al. 1992). This process is less common in rats. Loss of the normal lobular architecture with irregular Occasionally large proliferative hepatocellular lesions are growth pattern. observed that are difficult to diagnose. These lesions can com- Liver plates often impinge obliquely on surrounding press adjacent parenchyma and/or bulge from the surface. liver parenchyma. They also have, at least in some areas, normal to slightly dis- Hepatocytes with varying size and tinctorial staining torted lobular architecture with central veins and portal triads. pattern occur. The biologic nature of these lesions is unknown but because of Adenomas may also be classified morphology by their size and distinct compression, they are sometimes tinctorial and other cytoplasmic characteristics as included in the category of hepatocellular adenoma. It is rec- described for foci of cellular alteration. ommended that large lesions of this type with some evidence Staining of tumor cells may resemble that of the sur- of lobular architecture and the presence of central veins and rounding liver parenchyma and be classified as portal areas be diagnosed as non-regenerative hepatocellular amphophilic. hyperplasia (described previously in this document). Usually single nodules but multiple adenomas may be present. Fibrous encapsulation may occur. Hepatocellular Carcinoma (Figures 135–140) An enveloped portal triad may occasionally be present. Synonyms: Adenocarcinoma, liver cell; carcinoma, hepatic Mitotic index may be increased. cell; carcinoma, hepatocellular; carcinoma, liver cell; hepa- Areas of cellular atypia may be present (pleomorphic toma, malignant; hepatocarcinoma; nodule, type B. nuclei, coarsely clumped chromatin, large nucleoli, increased nucleus to cytoplasm ratio, cytoplasmic Pathogenesis: Spontaneous and following treatment with hepa- basophilia, focal attempts at trabeculae formation as totoxins that are carcinogenic xenobiotics; with gene altera- in carcinomas). tions in genetically engineered mice. Cells within adenomas may reveal signs of degenerative processes such as intracytoplasmic-inclusions, Diagnostic features: hyaline bodies, or vacuoles. Sinusoids may be compressed or ectatic. Local infiltrating growth and/or lack of distinct Necrosis is usually absent. demarcation. Pseudoinvasion of blood vessels may be present. Marked cellular pleomorphism may occur. Lesions may be grossly visible and/or bulge from nat- Alterations of tinctorial staining patterns may occur. ural surfaces. Loss of normal lobular architecture. May occur in livers with foci of cellular alteration Vascular invasion or metastases may be observed. and hepatocellular carcinomas. Increased mitotic index possible. Bile ducts may be present. Differential diagnosis: Hemorrhage, necrosis, and extramedullary hematopoietic foci may be present. Focus of cellular alteration—liver plates merge May occur as a single morphologic type or a combi- imperceptibly with surrounding hepatic parenchyma; nation of them as in the following. normal lobular architecture present. May metastasize to lung. Regenerative hyperplasia (in damaged liver)—evidence of prior or ongoing hepatocellular damage; nor- Trabecular: mal lobular architecture is present, albeit distorted. Non-regenerative hyperplasia—some evidence of Composed of well-differentiated hepatocytes form- lobular pattern and presence of central veins and por- ing trabeculae of multiple cell layers. tal triads. The plates alternate with sinusoids. Hepatocellular carcinoma—invasive growth; no Sometimes the sinusoids may be dilated forming clear demarcation; loss of lobular plates, architecture, blood lakes.

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Acinar (synonym: glandular): The trabecular type of hepatocellular carcinoma is the most common form in rats and to a lesser extent in mice. Neoplastic hepatocytes form a generally single layer Hepatocellular carcinomas may appear to arise within preexist- around a central clear space. ing adenomas, especially in mice (Figures 139 and 140). While The acinar structure may vary from tiny to huge cysts. some authors refer to these lesions as focus of carcinoma in Glandular pattern rarely involves more than 50% of adenoma, such lesions should be diagnosed as hepatocellular the neoplasm. carcinoma. These carcinomas exhibit the same morphological Acinar portion is interspersed with areas that have appearance as the carcinomas described earlier and hint toward either trabecular or solid architecture. a progression of adenoma into carcinoma. Attention should be paid to hemorrhagic areas, where proliferation of endothelial Solid: cells or formation of large sinusoidal-like spaces may lead to the erroneous diagnosis of a vascular tumor.

Cells tend to be poorly differentiated, small, hyperchromatic, and pleomorphic. Hepatoblastoma (Figures 141–144) Sometimes predominant cells are spindle shaped. Synonyms: Tumor, mixed, poorly differentiated. Single nuclei or multinucleated giant forms. Mitotic figures may be numerous and bizarre. The stroma is generally inconspicuous. Pathogenesis: Unknown but origin from liver blastema cells, The vascular structures are immature and often neoplastic hepatocytes, oval cells, and biliary epithelial cells thrombosed. proposed.

Adenoid (mouse): Diagnostic features:

Well-circumscribed nodule. A layer of neoplastic cells surrounds luminal structures. Distinct encapsulation with variable structure may be Lining cells usually consist of strongly basophilic present. cuboidal cells. Organoid structures are lined by vascular cavities filled with blood. Differential diagnosis: The channels are surrounded by several layers of tumor cells. Focus of cellular alteration—liver plates merge The cells are arranged either radially or concentrically imperceptibly with surrounding hepatic parenchyma; forming rosettes, trabeculae, or pseudo-glandular normal lobular architecture present. structures. Regenerative hyperplasia—evidence of prior or The center of the rosettes sometimes contains ampho- ongoing hepatocyte damage; normal lobular architec- philic material or small, endothelium-lined vessel. ture present, albeit distorted; demarcated from sur- Cells are small, strongly basophilic, and elongated. rounding liver tissue. Sometimes they may be more or less eosinophilic and Non-regenerative hyperplasia—some evidence of have smaller, rounder, and less hyperchromatic nuclei. lobular pattern and presence of central veins and por- Numerous mitotic figures are present. tal triads. Areas of large hemorrhage, pigmentation, fibrosis, or Hepatocellular adenoma—no local invasiveness, less necrosis are present. cellular atypia, sharply demarcated from surrounding Osteoid, bone, squamous differentiation, and extra- liver parenchyma. No formation of a trabecular pattern. medullary hematopoietic foci may be present. Cholangiocarcinoma—mucus may be present within the adenoid structures. If there is loss of mucus it is difficult to distinguish. Differential diagnosis: Hemangiosarcoma—proliferation of atypical endothelial cells forming blood spaces. Cholangiocarcinoma (poorly differentiated)—glandular structures with mucus production are present. Comment: Occur spontaneously with increased incidence in Often evidence is present of extensive fibrosis, but older rodents and following treatment with carcinogenic and not of osteoid, or bone formation. hepatotoxic xenobiotics (Bannasch and Zerban 1990; Brooks and Sarcoma, NOS—only mesenchymal structures are Roe 1985; Greaves and Faccini 1984; Jones and Butler 1978; present. Ward 1981; Harada et al. 1999; Eustis et al. 1990; Popp 1984, Carcinoma, hepatocellular-only sparse mesenchymal 1985; Vesselinovitch, Mihailovich, and Rao 1978). Diagnosis structures are present. Hepatic cell differentiation is may be modified based on growth pattern (Frith and Wiley 1982). obvious.

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Comment: Hepatoblastomas consist of organoid structures Hyperplasia, bile duct—multifocal, usually widespread. often oriented around vascular spaces (Harada et al. 1999; Cholangiofibrosis—central portions become Nonoyama et al. 1986, 1988; Turusov, Day, et al. 1973; atrophic, collagenized, and avascular whereas the Turusov, Deringer, et al. 1973). The cells are primitive in actively proliferating portion is at the periphery; appearance of scant, pale basophilic cytoplasm and ovoid mucus production is common. hyperchromatic nuclei. They are usually seen with other types Cholangiocarcinoma—proliferation of atypical cells, of hepatocellular tumors, especially within hepatocellular invasive growth. adenomas. Rare reports of this lesion in rats appear in literature. They are seen in certain strains of mice and have also Comment: Cholangioma is rare in control and treated been induced by certain hepatocarcinogens (Diwan, Ward, rodents (Bannasch and Zerban 1990; Brooks and Roe 1985; and Rice 1989). Frith and Ward 1979; Greaves and Faccini 1984; Harada Hepatoblastoma is generally seen within or adjacent to et al. 1999; Jones and Butler 1978; Lewis 1984; Maronpot hepatocellular neoplasms. In these cases, some preference has et al. 1986). been expressed for a single diagnosis of hepatoblastoma, rather than two diagnoses (the hepatoblastoma and the hepatocellular Cholangiocarcinoma (Figures 147 and 148) neoplasm). If this preferred convention of using the single diagnosis of hepatoblastoma is not used, then the alternative Synonyms: Adenocarcinoma, bile duct; adenocarcinoma, convention will need to be defined by the pathologist. Hepato- cholangiocellular; carcinoma, bile duct; carcinoma, cholangio- blastomas can have high rates of lung metastases in some cellular; cholangioma, malignant. experiments. Pathogenesis: Arise from proliferating cholangial cells.

Cholangioma (Figures 145 and 146) Diagnostic features:

Synonyms: Adenoma, bile duct; adenoma, biliary; adenoma, Biliary structures are usually glandular but may have cholangiocellular; cholangioma, benign. solid or papillary areas, may be poor in or free of mucus, and mostly have a minimal connective tissue Pathogenesis: Proliferation of biliary cells. component. Diagnostic features: Glandular lining cells are hyperbasophilic and have large nuclei and nucleoli but occasionally have clear Glandular acini may vary in size and shape. cytoplasm due to accumulated glycogen. Expansively growing with compression. Glands lined by single or multilayered cuboidal or Nucleus is round or oval occasionally vesicular with cylindrical cells. one or two conspicuous nucleoli. Clear-cut invasion into vascular and lymphatic struc- Cytoplasm is somewhat basophilic. tures and surrounding parenchyma may be present. Two types can be distinguished: May appear as a large solid mass. Evidence of metastasis or likelihood of metastasis is Simple: expected.

Generally uniform well-circumscribed neoplasm. Differential diagnosis: Acini are lined by a single layer of cuboidal cells varying in size. Cholangiofibrosis—central portions become Occasionally the cells are multilayered. atrophic, collagenized, and avascular whereas the Sparse vascular stroma may occur. actively proliferating portion is at the periphery; dilated glands with mucus production and accompa- Cystic: nying inflammation is common. Connective tissue response maybe pronounced. Characteristically composed of dilated glandular acini. Cholangioma—no invasion, sparse stroma, more uni- The acini are lined by cuboidal epithelium. form cells, single layer of cuboidal cells, may have Papillary structures are occasionally observed, pro- cystic glands. jecting into the lumen of the cysts. Carcinoma, hepatocellular (acinar)—no mucus pro- Clumps of liver cells may be seen between the cysts. duction; contains obvious neoplastic hepatocytes.

Differential diagnosis: Comment: Cholangiomas and cholangiocarcinomas are rarely seen as spontaneous neoplasms in rats and mice but may occur Bile duct cyst—lined by flattened epithelium; no expan- following exposure to hepatotoxic xenobiotics (Eustis et al. sive growth; not forming acini or papillary structures. 1990; Frith and Ward 1979; Harada et al. 1999; Jones and

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Butler 1978; Lewis 1984; Narama et al. 2003). A specific Carcinoma, hepatocholangiocellular—features of phenotype of cholangiocarcinoma with features of hepatocellular carcinoma and cholangiocarcinoma cholangiofibrosis consisting of dilated biliary glands, mucus are present. Hepatocytes arranged in nests, solid, tra- production, intestinal metaplasia, inflammatory cell infil- becular, or glandular patterns. Stratification and aty- trates, and fibrosis has been diagnosed in rats treated with a pia of biliary epithelial component is present. variety of hepatotoxic xenobiotics (Bannasch and Zerban 1990; Bannasch, Brenner, and Zerban 1985; Sirica 1992; Comment: Rare spontaneous lesion. A proliferative mixture of Kimbrough and Linder 1974; Maronpot et al. 1986). The hepatocytes and intrahepatic bile duct epithelium with neither distinction between cholangiofibrosis and this phenotype of component being malignant comprise to this tumor type cholangiocarcinoma is difficult and controversial and is pri- (Deschl et al. 2001; Frith and Ward 1979; Harada et al. marily based on extent of liver involvement since unequivocal 1999; Narama et al. 2003). metastasis is rare. These rare lesions of cholangiocarcinoma can contain para- Carcinoma, Hepatocholangiocellular (Figures 151 and 152) meters of cholangiofibrosis but show less inflammation and less mucus cyst(s) but with atypical ductular structures and can Pathogenesis: Proliferation of admixture of hepatocytes and metastasize. It has been proposed to diagnose this specific form intrahepatic bile duct epithelium. Stem cell origin speculated. of cholangiocarcinoma as ‘‘cholangiocarcinoma, intestinal Diagnostic features: type’’ (Greaves 2007), but that nomenclature as a separate (sub-) diagnosis was not favorably encouraged at a recent Features of hepatocellular carcinoma and cholangio- scientific workshop (NTP Satellite Symposium 2010). carcinoma are present. The hepatocytic component may be arranged in trabecular, glandular, or solid patterns. Adenoma, Hepatocholangiocellular (Figures 149 and 150) The biliary component may form acini or small nests Pathogenesis: Proliferation of admixture of hepatocytes and without lumen. Stratification or cellular atypia may intrahepatic bile duct epithelium. Stem cell origin speculated. occur. Occasionally ducts lined by both cell types, hepato- Diagnostic features: cytic and biliary epithelial cells, may be present. Areas of hemorrhage and necrosis may be present. Features of hepatocellular adenoma and cholangioma Mitotic figures may be numerous. are present. Areas composed of cords of neoplastic hepatocellular Differential diagnosis: cells merge with areas composed of ducts lined by neoplastic epithelium that resembles bile duct epithe- Carcinoma, hepatocellular—composed of malignant lium. Hepatocytes may be seen forming ductules or hepatocytes. ducts. Cholangiocarcinoma—composed of malignant duct The neoplastic biliary epithelium forms slightly epithelium. dilated acini lined by cuboidal cells. Adenoma, hepatocholangiocellular—features of Stratification and atypia of the biliary epithelium is hepatocellular adenoma and cholangioma are pres- minimal or absent. ent. Hepatocytic component has typical cord-like Stromal component may be absent. arrangement of cells. Evidence of stratification and Hepatocytes may have some alteration in staining atypia of biliary epithelial component is minimal or (eosinophilic, basophilic, or clear cell) compared to absent. surrounding parenchyma. Mitotic figures are rare. Comment: Rare spontaneous lesion. This neoplasm contains neoplastic elements of both hepatocytes and bile duct epithe- Differential diagnosis: lium (Deschl et al. 2001; Frith and Ward 1979; Harada et al. 1999; Narama et al. 2003; Teredesai, Wohrmann, and Schlage Adenoma, hepatocellular—composed of neoplastic 2002). A diagnosis of malignancy may be based on just one of hepatocytes. Loss of normal lobular architecture with the components being malignant. irregular growth pattern. Liver plates often impinge perpendicular or obliquely on the surrounding par- Tumor, Ito Cell, Benign (Figures 153 and 154) enchyma. Distinct compression is present. Cholangioma—composed of neoplastic duct Synonyms: Fat-storing cell tumor; stellate cell tumor, lipoma. epithelium. Well demarcated; usually solitary. The acini are lined by cuboidal uniform and well- Pathogenesis: Arises from fat-storing perisinusoidal cells, differentiated epithelium. so-called Ito cells.

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Diagnostic features: Atypical cells are sparse, pleomorphism is usually absent, and mitotic figures may be numerous. Unicentric or multicentric unencapsulated mass. The tumors grow along sinusoids and vessels and Focal or diffuse accumulation of tumor cells. frequently involve other organs such as the lung and Partially distinct compression of adjacent hepatic spleen. parenchyma. Metastases and spread on serosal surfaces and in the May grow in sheets, clusters, or along cords of vascular spaces are common. hepatocytes. Minimal fibrosis may be present. Cells vary in size and shape and are vacuolated. Multiple cytoplasmic fat droplets of different size Differential diagnosis: occur. The nuclei are ovoid or round and may be indented by Malignant fibrous histiocytoma—the tumor has a cytoplasmic lipid droplets. mixed cell population of histiocyte-like cells, Frequently evidence of atrophy of adjacent liver tis- bizarre tumor giant cell, fibroblasts, and undifferen- sue is present. tiated cells. The fibrous component is always Modest amount of collagenous matrix may be prominent. present. Malignant lymphoma—no giant cells are seen and At the margin of the lesion, spindle-shaped cells are lymph nodes and spleen are frequently involved. His- frequently observed. tiocytic sarcoma and lymphoma may occur together Unencapsulated. in the liver.

Differential diagnosis: Comment: Histiocytic sarcomas occur at a low frequency in rats and mice (Harada et al. 1999; Eustis et al. 1990). The tumor Ito cell hyperplasia—multicentric lipomatous can be part of a systemic lesion involving various tissues lesion or a singular small lesion that does not (spleen, lung, and uterus); when involving only the liver it is show distinct compression of surrounding liver sometimes referred to as Kupffer cell sarcoma (Deschl et al. parenchyma. 2001; Carlton et al. 1992). Liposarcoma—various types of fat cells, foam cells, giant cells, myxoid cells, or fibroblast-like cells may be present. Hemangioma (Figures 156 and 157) Synonym: Hemangioendothelioma, benign. Comment: Ito cell neoplasms are extremely rare. As a consequence, the histogenesis and the biological behavior of these Pathogenesis: Arises from endothelial cells lining vascular tumors are not well established (Dixon et al. 1994; Enzan spaces, most commonly of the hepatic sinusoids. 1985; Tillmann et al. 1997). Diagnostic features:

Histiocytic Sarcoma (Figure 155) A moderate compression of the surrounding tissues is usually seen. Synonym: Kupffer cell sarcoma. The tumor is rarely encapsulated. Blood-filled spaces lined with a single layer of Pathogenesis: May arise from fixed macrophages (Kupffer prominent uniform endothelial cells without cells) attached to the sinusoidal endothelial cells or from circu- atypia. lating macrophages, unless metastasized from other organs Mitotic figures are rarely present. (e.g., skin, uterus). Solid cellular areas of uniform cells without atypia Diagnostic features: may occur. Often multifocal in the liver or in livers with angiec- Characteristically form nodules within the liver tasis of mice. that may contain a central area of necrosis surrounded by palisaded tumor cells (multicentric Capillary type: origin). Uniform population of rounded or oval cells with Closely packed capillary structures. foamy, eosinophilic cytoplasm, indistinct cell bound- Minimal stroma between the vascular spaces. aries, and elongated or folded nuclei. Cavernous type: Sometimes are present multinucleated giant cells of Large vascular channels. foreign body scattered throughout the tumor. Abundant connective tissue between the larger channels.

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Differential diagnosis: collagen bundles, and surfaces with no cytological and histological features of malignancy. Angiectasis—dilated vessels or sinusoids are not Hemangioma—no cytological and histological fea- increased in number and have normal structure and tures of malignancy such as cellular pleomorphism, well-differentiated endothelial cells. increased mitotic activity, tissue invasion, or metas- Hyperplasia, angiomatous—the hyperplastic vessels tases are present. are lined by a normal endothelium and cause no or Hemangiopericytoma, malignant—the tumor only minimal compression of the surrounding consists of tightly packed spindle-shaped tumor tissues. cells that have cytological features of malignancy Lymphangioma—the vascular spaces are devoid of and encircle thin-walled vascular channels erythrocytes. (‘‘fingerprints’’). Hemangiosarcoma—cytological and histological Fibrosarcoma—the tumor lacks a distinct vascular features of malignancy are present, such as cellular pattern with prominent endothelial cells. pleomorphism, increased mitotic activity, tissue invasion, or metastases. Comment: As in the case of hemangiomas, the occurrence of Hemangiopericytoma, benign—the tumor consists of hemangiosarcomas in rodents has been well described in the tightly packed spindle-shaped tumor cells encircling published literature (Binhazim, Coghlan, and Walker 1994; thin-walled vascular channels (‘‘fingerprints’’). In Booth and Sundberg 1996; Faccini, Abbott, and Paulus reticulin-stained sections, a dense reticulin meshwork 1990; Frith and Ward 1988; Frith and Wiley 1982; Giddens surrounds individual tumor cells. and Renne 1985; Greaves and Barsoum 1990; Greaves and Faccini 1984; Heider and Eustis 1994; Jones and Butler Comment: The occurrence of hemangiomas in rodents has 1975; Maita et al. 1988; Mitsumori 1990; Morgan et al. been well documented (Booth and Sundberg 1996; Carter 1984; Peckham and Heider 1999; Popper, Maltoni, and 1973; Faccini, Abbott, and Paulus 1990; Frith and Ward Selikoff 1981; Pozharisski and Turusov 1991; Sakamoto, 1988; Frith and Wiley 1982; Heider and Eustis 1994; Jones Takayama, and Hosoda 1989; Solleveld et al. 1988; Stewart and Butler 1975; Maita et al. 1988; Greaves and Barsoum 1979; Yamate et al. 1988). 1990; Greaves and Faccini 1984; Mitsumori 1990; Peckham Endothelial cell-derived hemangiosarcomas can be and Heider 1999; Stewart 1979; Stewart et al. 1980; Squire induced in rats and mice by a wide range of industrial, nat- and Levitt 1975; Ward et al. 1979; Yamate et al. 1988; ural, and pharmaceutical compounds. There are numerous Zwicker et al. 1995). The cavernous type of hemangioma is examples documenting the progress that is being made in considered by some authors to be a congenital malformation recent years in suggesting the genesis and potential rele- rather than a neoplasm. vance for human risk assessment of these tumors (Klaunig and Kamendulis 2005; Laifenfeld et al. 2010; Ohnishi Hemangiosarcoma (Figures 158-160) et al. 2007). Synonym: Hemangioendothelioma, malignant.

Pathogenesis: Arises from pluripotential mesenchymal stem H. Other Liver Lesions cells; endothelial cells of blood vessels or hepatic sinusoids. Extramedullary Hematopoiesis (Figures 161 and 162) Diagnostic features: Synonym: Hematopoietic cell proliferation; myelopoiesis; The endothelial lining cells have a moderate erythropoiesis. pleomorphism. Endothelial cells may be multilayered and/or Pathogenesis: In adult rodent, a response to increased hemato- clustered. poietic demand. Various vascular patterns may be present, but vessels are not well formed. Diagnostic features: Undifferentiated or fibrosarcomatous areas may also be seen. Small aggregates of hematopoietic cells are randomly Mitotic figures are often present. distributed in the hepatic sinusoids as well as around Local invasion and metastases are often present. central veins and in portal areas. Both erythyroid and granulocytic cells may be pres- Differential diagnosis: ent in the aggregates; rarely megakaryocytes may be present. Granulation tissue—the newly formed blood vessels Typically not associated with hepatocellular necrosis are typically arranged perpendicular to fibroblasts, or degeneration.

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Differential diagnosis: Pancreatic Acinar Metaplasia (Figures 164 and 165) Pathogenesis: Islands of pancreatic tissue localized within the Mononuclear cell aggregates—lymphocytes and his- hepatic parenchyma are rare spontaneous occurrences in rats tiocytic cells present alone or in addition to mature but have been reported following prolonged exposures to poly- myeloid cells. chlorinated biphenyls (Kimbrough 1973; Eustis et al. 1990; Focal inflammation—mixed mature leukocytes, Greaves 2007). often associated with or a response to cellular necrosis. Diagnostic features:

The islands of pancreatic tissue resemble normal aci- Comment: Extramedullary hematopoiesis (EMH) can be nar pancreas with zymogen granules. observed in rodent liver occasionally in response to an An integrated component of the hepatic tissue. increased hematopoietic demand. Hematopoiesis is normally found in the embryonic liver where embryonic hematopoiesis Differential diagnosis: dramatically expands at mid-gestation but decreases after birth. Hepatic EMH is seen more common in rodents than in man, Artifact during tissue processing—tissue ‘‘floater.’’ more common in mice than rats, and more often observed in Metastatic pancreatic acinar neoplasia—locally inva- females as compared to males as a general rule (Eustis et al. sive with tissue destruction; presence of a primary 1990; Harada et al. 1996, 1999). Precipitating factors for the pancreatic acinar neoplasm in the pancreas. occurrence are: anemia, stress, xenobiotic toxicity, infection, Ectopic pancreatic tissue—a collection of pancreatic neoplasia (e.g., histiocytic sarcoma), and pregnancy. When the tissue adjacent to but not integrated within hepatic erythroid precursors predominate, often the term extramedul- parenchyma. lary erythropoiesis is used. Comment: In spontaneous cases, a distinction between metaplasia and ectopic pancreas may not be possible. Since both Intrahepatocellular Erythrocytes (Figure 163) pancreas and liver are embryologically related, there is a defi- Synonyms: Emperipolesis, cytoplasmic inclusions; hepatic nite potential for metaplasia. erythrophagocytosis. Hepatocytes, Glandular Metaplasia (Figures 166 and 167) Pathogenesis: Unknown. Pathogenesis: Proliferation of hepatocytes to form glandular structures. Diagnostic features: Diagnostic features: Individual or small clusters of enlarged hepatocytes containing intact erythrocytes. A few to numerous glandular structures diffusely Affected hepatocytes are markedly enlarged. scattered in the hepatic parenchyma. Marginated hepatocyte nucleus. May also be present in hyperplastic nodules and hepatocellular adenomas. Differential diagnosis: Vary in size from one to ten times the diameter of a portal bile duct. Angiectasis—dilated vascular spaces lined by Lining cells resemble hepatocytes but smaller more endothelial cells; dilated vascular channels and cuboidal glandular cells resemble biliary epithelium. spaces frequently contain erythrocytes. Glandular lumen may contain granular eosinophilic Cystic degeneration—consists of enlarged stellate material and sometime free blood cells. cells with flocculent eosinophilic cytoplasm. Differential diagnosis: Comment: The intracytoplasmic inclusion of large numbers of erythrocytes in hepatocytes has been seen exclusively in mice Cholangioma; glandular acini may vary in size and (Harada et al. 1999). It has occurred in at least nine separate shape, expansively growing with compression. cancer bioassays and one fourteen-day study in B6C3F1 mice. Hepatocholangioma; features of hepatocellular ade- In two of these studies it appears to have been exacerbated or noma and cholangioma are present. possibly caused by treatment. Attempts to demonstrate active erythrophagocytosis by electron microscopy have been unsuc- Comment: Partial replacement of hepatic parenchyma by cessful. A potential mechanism is emperiopolesis. Internaliza- glandular structures with features resembling hepatocytes has tion of erythrocytes in hepatocytes has been reported in been observed in chronic studies of 3, 3’, 4, 4’, 5- hibernating frogs (Barni and Bernocchi 1991). pentachlorobiphenyl and 2, 3’, 4, 4’, 5-pentachlorobiphenyl

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(NTP Toxicology and Carcinogenesis Studies 2006). It is Diagnostic features: speculated that the glandular structures represent abnormal differentiation of hepatic precursor cells (NTP Toxicology and Mature pancreatic acinar tissue located in wall of the Carcinogenesis Studies 2006). gallbladder (Harada et al. 1999).

Intravascular Hepatocytes (Figure 168) Differential diagnosis:

Synonyms: Vascular pseudoinvasion; vascular infiltration of Metastatic pancreatic acinar carcinoma—locally hepatocytes. invasive neoplasm with distortion and/or destruction of gallbaldder tissue. Pathogenesis: Unknown. Sporadic occurrence. Pancreatic metaplasia—smoothly integrated normal Diagnostic features: appearing pancreatic tissue within the gallbladder mucosa. Protrusion of normal appearing hepatocytes into hepatic veins and within the contour of the vessel. Degenerative Lesions Usually involves medium to large size hepatic veins. Infiltrating hepatocytes are covered by an endothelial Hyalinosis, Gallbladder (Figures 171–174) cell lining. Synonyms: Hyalinosis, cytoplasmic inclusions, crystals.

Differential diagnosis: Pathogenesis: A change in the gallbladder epithelium that can be induced by inflammation and unknown factors. Extension of perivascular focus of cellular alteration, usually basophilic. Diagnostic features: Metastatic hepatocellular carcinoma. The gallbladder epithelial cells contain a hyaline Comment: Intravascular infiltration of hepatocytes is rarely cytoplasm, which is uniformly eosinophilic. seen in control and treated mice. A similar change was The protein is usually immunoreactive for Ym1/ reported in diethylnitrosamine treated mice as part of a baso- Ym2. philic focus of cellular alteration response (Goldfarb et al. Epithelial cells may contain eosinophilic needle-like 1983; Koen, Pugh, and Goldfarb 1983). The significance of crystals and the same and larger crystals may be seen this change is unknown. extracellularly. Maybe associated with hyalinosis in other tissues, such as bile duct epithelium, stomach, and lung. I. Gallbladder Lesions Differential diagnosis: Congenital Lesions Heterotopic Hepatocytes Other degenerative cellular changes without hyali- nized eosinophilic cytoplasm. Pathogenesis: Developmental anomaly; postnatal Amyloidosis—pale eosinophilic extracellular deposits. transdifferentiation. Diagnostic features: Comment: The hyaline protein in the cells has been shown to be Ym1/Ym2 (now Chi313), a chitinase-like protein, with Cells morphologically identical to mature hepato- unknown functions. In sickle cell mice, it is associated with cytes are present in the submucosa of the gallbladder gallstones. Hyalinosis is rare in most lines of mice (Harada (Harada et al. 1999). et al. 1999; Hsu et al. 2006; Yang and Campbell 1964) but may occur in high incidence in 129 and B6;129 mice (Ward et al. Differential diagnosis: 2001) and in some genetically engineered mouse lines. Hyali- nosis is reported to occur in increased incidence in B6C3F1 Metastatic hepatocellular neoplasm—locally inva- female mice exposed to penicillin. sive expansile mass of atypical hepatocytes. Artifact—tissue ‘‘floater.’’ Glandular Metaplasia Synonym: Adenomatoid change. Heterotopic Acinar Pancreas (Figures 169 and 170) Pathogenesis: Developmental abnormality; postnatal Pathogenesis: Spontaneous and associated with inflammatory transdifferentiation. and proliferative changes in gallbladder.

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Diagnostic features: Comment: Gallstones are rare spontaneously in mice but may be experimentally induced by various methods (Chang, Thickened mucosa with diffuse or focal proliferation Suh, and Kwon 1999; Hsu et al. 2006; Ichikawa et al. 2009; Lee of tall columnar cells forming numerous glands in the and Scott 1982; Lewis 1984; Rege and Prystowsky 1998; lamina propria. Tepperman, Caldwell, and Tepperman 1964; Trotman et al. The gallbladder epithelial cells may show increased 1983; Xie et al. 2009). cell proliferation. Hypertrophic columnar cells with uniform, homoge- Inflammatory Lesions neous, bright eosinophilic cytoplasm forming glandular structures with lumen containing eosinophilic Cholecystitis (Figures 177 and 178) crystals. Synonym: Inflammation, gallbladder. Chronic inflammation of the gallbladder may be present. Pathogenesis: Toxicant exposure and bacterial and viral infections (Greaves 2007; Harada et al. 1999). Comment: Glandular metaplasia has been observed at low pre- Diagnostic features: valence in the gall bladder and intra-hepatic bile ducts (all associated with cholelithiasis), cholecystitis, cholangitis, May be accompanied by ulcers or erosions of the papillomatous hyperplasia, papilloma, intra-mural cysts, and mucosal lining cells. focal epithelial ulceration of aged mice from life span carcino- Types of inflammation range from acute to chronic, genicity studies. The lesions are found predominantly in female including a granulomatous reaction. mice (Lewis 1984) and can occur spontaneously in some strains Lumen may contain necrotic cellular debris. of mice. Other metaplastic changes have also been described in Mucosal hyperplasia and mucinous metaplasia may the human gallbladder, including goblet cells, paneth cells, be present in some cases of inflammation. and/or enterochromaffin cells in the mucosa (Hruban, Argani, Initial change may be submucosal edema. and Ali 2006).

Proliferative Lesions Gallbladder, Calculi (Figures 175 and 176) Hyperplasia, Gallbladder (Figures 179 and 180) Synonyms: Stones, gallstones, choleliths. Pathogenesis: Irritation of gallbaldder mucosa and after xenobiotic exposure. Pathogenesis: Excess dietary factors and altered metabolism. Diagnostic features: Diagnostic features: Lesion is often small. Grossly visible concretion(s) in the gallbladder of Lesion varies from a few cells on papillary folds to mice. small papillary projections. Grossly, may be solid or soft, single or multiple, and Epithelium is usually single layered. of various colors including white and pigmented (yel- Cells are well differentiated. low, grey). Minimal atypia may be present. Depending on the etiology, the stone may be composed of a mixture of cholesterol, calcium salts, Differential diagnosis: hemoglobin, and occasionally as a pure stone composed of just one of these substances. Adenoma—growth pattern is disordered. May exhi- Often associated with inflammatory lesions of the bit some atypia gallbladder. Adenocarcinoma—increased cellular atypia. Disor- dered growth pattern. Invasive growth. Differential diagnosis: Adenomatoid change/glandular metaplasia—focal or diffuse proliferation of epithelial cells forming gland- Mineralization—associated with necrosis, necrotic ular structures. Cells are well differentiated, usually benign or malignant tumor, and inflammatory columnar, and eosinophilic, with little or no cellular conditions. atypia. Distinct eosinophilic crystals are present in Neoplasia (Gross) —histologically, it is neoplastic. cytoplasm, or in lumen of glands. Inflammation—inflammatory exudates may be seen without gallstone formation. Comment: Details related to gallbladder hyperplasia can be Parasites—can be seen histologically as parasites. found in several references (Deschl et al. 2001; Harada et al.

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1996, 1999; Yoshitomi, Alison, and Boorman 1986; Yoshitomi et al. 2001; Harada et al. 1996, 1999; Lewis 1984; Yoshitomi, and Boorman 1994). Alison, and Boorman 1986; Yoshitomi and Boorman 1994).

Adenoma, Gallbladder (Figures 181–183) Adenocarcinoma, Gallbladder Synonym: Adenoma, papillary. Pathogenesis: Arises from epithelium of the gallbladder. Diagnostic features: Pathogenesis: Arises from epithelium of gallbladder. Diagnostic features: May be a sessile broad-based mass or characterized by diffusely thickening of mucosa. In general well differentiated and solitary. Growth pattern is disordered. Growth is disordered, predominantly papillary- or Cellular atypia is present. cauliflower-like. Cytoplasm is scant and basophilic. Epithelium is single layered, but occasionally may be Nuclei are enlarged. multilayered. Mitotic figures are common. Amount of fibrovascular stroma is variable. Invasion of the wall of the gallbladder or of adjacent Cells may exhibit some atypia, with enlarged nuclei tissue. or even giant nuclei with one or two nucleoli. Mitotic figures may be present. Differential diagnosis: Often inflammatory cellular infiltration and focal mineralization of the stroma may be present. Hyperplasia—lesion is small. Exhibits only little evidence of atypia. Differential diagnosis: Adenoma—no evidence of invasive growth. Less cellular atypia. Hyperplasia (focal)—small lesion. Exhibits only lit- Adenomatoid change (glandular metaplasia)— tle evidence of atypia. focal or diffuse proliferation of epithelial cells Adenocarcinoma—increased cellular atypia. Disor- forming glandular structures. Cells are well differ- dered growth pattern. Invasive growth. entiated, usually columnar, and eosinophilic, with Adenomatoid change/glandular metaplasia—focal or little or no cellular atypia. Distinct eosinophilic diffuse proliferation of epithelial cells forming gland- crystals are present in cytoplasm, or in lumen of ular structures. Cells are well differentiated, usually glands. columnar, and eosinophilic, with little or no cellular atypia. Distinct eosinophilic crystals are present in Comment: Benign and malignant epithelial neoplasms of the cytoplasm, or in lumen of glands. gallbladder are described in several published references (Deschl et al. 2001; Harada et al. 1996, 1999; Lewis 1984; Comment: Benign and malignant epithelial neoplasms of the Yoshitomi, Alison, and Boorman 1986; Yoshitomi and gallbladder are described in several published references (Deschl Boorman 1994).

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FIGURE 1.—Gross appearance and tissue trimming recommendations for a normal rodent liver. Ref. to http://reni.item.fraunhofer.de/reni/trimming/ index.php. FIGURE 2.— Two-dimensional microarchitecture of the liver. FIGURE 3.—Rat liver. Hepatodiaphragmatic nodule. FIGURE 4.—Rat liver. Hepatodiaphragmatic nodule with intranuclear inclusions (chromatin). Higher magnification of Figure 3. FIGURE 5.—Rat liver. Macrovesicular fatty change. FIGURE 6.—Rat liver. Macrovesicular fatty change. Higher magnification of Figure 5.

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FIGURE 7.—Mouse liver. Fatty change, microvesicular. FIGURE 8.—Mouse liver. Mixture of fatty change and cytoplasmic glycogen. FIGURE 9.— Mouse liver. Mixture of fatty change and cytoplasmic glycogen. Higher magnification of Figure 8. FIGURE 10.—Mouse liver. Tension lipidosis. FIGURE 11.—Mouse liver. Tension lipidosis. Higher magnification of Figure 10. FIGURE 12.—Rat liver. Focal fatty change.

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FIGURE 13.—Rat liver. Focal fatty change. Higher magnification of Figure 12. FIGURE 14.—Rat liver. Phospholipidosis. FIGURE 15.—Rat liver. Phospholipidosis. Higher magnification of Figure 14. FIGURE 16.—Rat liver. Phospholipidosis. EM concentric membrane bound lysosomal myeloid bodies/lamellar bodies. FIGURE 17.—Rat liver. Phospholipidosis. Central microvesiculation; positive LAMP-2 staining. FIGURE 18.—Mouse liver. Amyloidosis.

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FIGURE 19.—Mouse liver. Amyloidosis. Higher magnification of Figure 18. FIGURE 20.—Mouse liver. Focal mineralization associated with centrilobular necrosis. FIGURE 21.—Mouse liver. Pigment deposition in a focus of histiocytes. FIGURE 22.—Rat liver. Pigmentation. FIGURE 23.—Mouse liver. Centrilobular hypertrophy with cholestasis. FIGURE 24—Mouse liver. Cholestasis.

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FIGURE 25.—Rat liver. Pigment in hypertrophic hepatocytes consistent with bile. Cholestasis. FIGURE 26.—Mouse liver. Hyalinosis of hyperplastic bile ducts with crystal formation and peribiliary inflammatory cell infiltrate. FIGURE 27.—Mouse liver. Hyalinosis of hyperplastic bile ducts with crystal formation and peribiliary inflammatory cell infiltrate. Higher magnification of Figure 26. FIGURE 28.—Mouse liver. Hyalinosis of hyperplastic bile ducts with crystal formation. FIGURE 29.—Mouse liver. Numerous intracytoplasmic hyaline bodies in an hepatocellular adenoma. FIGURE 30.—Mouse liver. Intranuclear inclusion body, cytoplasmic invagination.

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FIGURE 31.—Mouse liver. Intranuclear inclusion bodies. FIGURE 32.—Mouse liver. Cytoplasmic inclusions in an hepatocellular adenoma. FIGURE 33.—Mouse liver. Plasma influx. FIGURE 34.—Mouse liver. Centrilobular hepatocellular hypertrophy. FIGURE 35.—Mouse liver. Higher magnification of centrilobular hepatocellular hypertrophy. FIGURE 36.—Mouse liver. Hepatocellular hypertrophy.

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FIGURE 37.—Mouse liver. Hepatocellular hypertrophy. FIGURE 38.—Mouse liver. Centrilobular hepatocellular hypertrophy. FIGURE 39.—Rat liver. Hepatocellular hypertrophy with eosinophilic granular cytoplasm following treatment with a peroxisome proliferating xenobiotic. FIGURE 40.—Rat liver. Hepatocellular hypertrophy following treatment with a peroxisome proliferating xenobiotic. FIGURE 41.—Rat liver. Hepatocyte enlargement confirmed as mitochondrial hypertrophy. FIGURE 42.—Rat liver. Atrophy.

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FIGURE 43.—Mouse liver. Hepatic atrophy. FIGURE 44.—Mouse liver. Cytoplasmic alteration. FIGURE 45.—Mouse liver. Hydropic degeneration. FIGURE 46.—Rat liver. Cystic degeneration. FIGURE 47.—Rat liver. Cystic degeneration. FIGURE 48.—Mouse liver. Apoptosis.

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FIGURE 49.—Mouse liver. Apoptosis. FIGURE 50.Mouse liver. Apoptosis. FIGURE 51 and 53 Mouse liver. Necrosis, focal. FIGURE 52. Mouse liver. Necrosis, multifocal. FIGURE 54. Rat liver. Bridging centrilobular necrosis.

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FIGURE 55.—Rat liver. Bridging centrilobular necrosis. Higher magnification of Figure 54. FIGURE 56.—Rat liver. Centrilobular necrosis with early bridging. FIGURE 57.—Rat liver. Centrilobular necrosis with early bridging. Higher magnification of Figure 56. FIGURE 58.—Rat liver. Centrilob- ular bridging necrosis with mineralization. FIGURE 59.—Rat liver. Centrilobular bridging necrosis with mineralization. Higher magnification of Figure 58. FIGURE 60.—Rat liver. Midzonal necrosis.

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FIGURE 61.—Rat liver. Midzonal necrosis. Higher magnification of Figure 60. FIGURE 62.—Mouse liver. Periportal necrosis. FIGURE 63.—Mouse liver. Diffuse necrosis with inflammation and bile duct hyperplasia in lower left of figure. FIGURE 64.—Mouse liver. Diffuse necrosis. Multinucleated giant cells in MHV infection. Higher magnification of Figure 63. FIGURE 65.—Mouse liver. Karyocytomegaly. FIGURE 66.—Mouse liver. Multinucleated hepatocytes. Karyocytomegaly.

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FIGURE 67.—Rat liver. Biliary cysts. FIGURE 68.—Rat liver. Biliary cysts. FIGURE 69.—Rat liver. Biliary cysts. Higher magnification of Figure 68. FIGURE 70.—Mouse liver. Generalized inflammation, postnecrotic mild fibrosis. Mouse hepatitis virus infection. FIGURE 71.—A descriptive approach for classifying inflammatory responses in the liver. FIGURE 72.—Mouse liver. Focal neutrophil infiltrate associated with hepatocyte necrosis.

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FIGURE 73.—Mouse liver. Multifocal mononuclear cell infiltrates. Norovirus infection. FIGURE 74.—Rat liver. Infiltration, mononuclear. Granu- lomatous inflammation with pigment-laden histiocytes and multinucleated giant cells. FIGURE 75.—Rat liver. Mixed inflammatory cell infiltrate and granulomatous inflammation. FIGURE 76.—Infiltration, mononuclear (microgranulomas). FIGURE 77.—Rat liver. Infiltration, mononuclear. FIGURE 78.—Rat liver. Infiltration, mononuclear. Granulomatous inflammation (cholesterol granuloma).

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FIGURE 79.—Rat liver. Infiltration, mononuclear. Granulomatous inflammation (cholesterol granuloma). Higher magnification of Figure 78. FIGURE 80.—Mouse liver. Focal neutrophil infiltrate. FIGURE 81.—Mouse liver. Periportal inflammatory cell infiltrate. FIGURE 82.—Rat liver. Fibrosis. Early bridging between lobules. FIGURE 83.—Rat liver. Fibrosis. Higher magnification of Figure 82. FIGURE 84.—Mouse liver. Silver stain demonstrating Helicobacter sp.

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FIGURE 85.—Rat liver. Chronic passive congestion. FIGURE 86.—Rat liver. Angiectasis. FIGURE 87.—Rat liver. Angiectasis. FIGURE 88.—Rat liver. Thrombosis with associated area of necrosis. FIGURE 89.—Mouse liver. Infarcted lobe. FIGURE 90.—Mouse liver. Infarct.

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FIGURE 91.—Rat liver. Endothelial karyomegaly (sinusoidal cell karyomegaly). FIGURE 92.—Mouse liver. Increased mitoses. FIGURE 93.—Rat liver. Diffuse basophilic focus. FIGURE 94.—Rat liver. Diffuse basophilic focus. Higher magnification of Figure 93. FIGURE 95.—Rat liver. Baso- philic (tigroid) focus of cellular alteration. FIGURE 96.—Rat liver. Basophilic focus of cellular alteration with cystic degeneration.

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FIGURE 97.—Rat liver. Basophilic focus of cellular alteration. FIGURE 98.—Rat liver. Eosinophilic focus of cellular alteration. FIGURE 99.—Rat liver. Eosinophilic focus of cellular alteration. Higher magnification of Figure 98. FIGURE 100.—Rat liver. Eosinophilic focus of cellular alteration with pale pink cytoplasm. FIGURE 101.—Mouse liver. Large basophilic focus of cellular alteration with glycogen present in centrally located hepatocytes. FIGURE 102.—Mouse liver. Large basophilic focus of cellular alteration with glycogen present in centrally located hepatocytes. Higher magnification of the edge of Figure 102.

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FIGURE 103.—Rat liver. Mixed cell foci. FIGURE 104.—Rat liver. Mixed cell focus. Higher magnification of Figure 103. FIGURE 105.—Mouse liver. Clear cell focus. FIGURE 106.—Mouse liver. Clear cell focus. Higher magnification of Figure 105. FIGURE 107.—Rat liver. Clear cell focus of cellular alteration. FIGURE 108.—Rat liver. Amphophilic focus.

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FIGURE 109.—Rat liver. Early non-regenerative hyperplasia. FIGURE 110.—Rat liver. Non-regenerative hyperplasia. FIGURE 111.—Rat liver. Regenerative hyperplasia. FIGURE 112.—Rat liver. Regenerative hyperplasia. Higher magnification of Figure 111. FIGURE 113.—Rat liver. Regen- erative hyperplasia. Higher magnification of Figure 111. FIGURE 114.—Mouse liver. Kupffer cell hyperplasia.

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FIGURE 115.—Mouse liver. Kupffer cell hyperplasia. FIGURE 116.—Mouse liver. Ito cell hyperplasia. FIGURE 117.—Mouse liver. Ito cell hyperplasia. Higher magnification of Figure 116. FIGURE 118.—Mouse liver. Ito cell hyperplasia, multifocal. FIGURE 119.—Rat liver. Bile duct hyperplasia. FIGURE 120.—Rat liver. Bile duct hyperplasia with associated mononuclear inflammatory cell infiltrate.

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FIGURE 121.—Rat liver. Bile duct hyperplasia with mononuclear inflammatory cell infiltration and early fibrosis. FIGURE 122.—Rat liver. Bile duct hyperplasia. FIGURE 123.—Rat liver. Cholangiofibrosis. FIGURE 124.—Rat liver. Cholangiofibrosis. FIGURE 125.—Rat liver. Cholangiofibro- sis. FIGURE 126.—Rat liver. Cholangiofibrosis.

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FIGURE 127.—Rat liver. Oval cell hyperplasia. FIGURE 128.—Mouse liver. Oval cell hyperplasia. FIGURE 129.—Mouse liver. Oval cell hyperplasia. FIGURE 130.—Mouse liver. Large hepatocellular adenoma. FIGURE 131.—Mouse liver. Hepatocellular adenoma, eosinophilic. FIGURE 132.— Mouse liver. Hepatocellular adenoma, eosinophilic. Higher magnification of Figure 131.

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FIGURE 133.—Mouse liver. Hepatocellular adenoma. FIGURE 134.—Mouse liver. Hepatocellular adenoma. High magnification of Figure 133. FIGURE 135.—Mouse liver. Hepatocellular carcinoma. FIGURE 136.—Mouse liver. Hepatocellular carcinoma. Prominent glandular formation. Higher mignification of Figure 135. FIGURE 137.—Mouse liver. Hepatocellular carcinoma. Prominent glandular and trabecular formation. Higher mignification of Figure 135. FIGURE 138.—Mouse liver. Hepatocellular carcinoma with trabecular and glandular formation.

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FIGURE 139.—Mouse liver. Hepatocellular carcinoma arising in an hepatocellular adenoma. FIGURE 140.—Mouse liver. Hepatocellular carcinoma arising in a hepatocellular adenoma. Higher magnification of Figure 139. FIGURE 141.—Mouse liver. Hepatoblastoma. FIGURE 142.—Mouse liver. Hepatoblastoma. Higher magnification of Figure 141. FIGURE 143.—Mouse liver. Hepatoblastoma. Osseous metaplasia is present. FIGURE 144.— Mouse liver. Hepatoblastoma. High magnification of Figure 143.

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FIGURE 145.—Mouse liver. Cholangioma. FIGURE 146.—Mouse liver. Cholangioma. Higher magnification of Figure 145. FIGURE 147.—Mouse liver. Cholangiocarcinoma. FIGURE 148.—Mouse liver. Cholangiocarcinoma. Higher magnification of Figure 147. FIGURE 149.—Rat liver. Hepatocholan- gioma (adenoma, hepatocholangial). FIGURE 150.—Rat liver. Hepatocholangioma (adenoma, hepatocholangial). Higher magnification of Figure 149.

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FIGURE 151.—Rat liver. Hepatocholangiocarcinoma (Carcinoma, hepatocholangial). FIGURE 152.—Rat liver. Hepatocholangiocarcinoma (Carcinoma, hepatocholagial). High magnification of Figure 151. FIGURE 153.—Mouse liver. Ito cell tumor. FIGURE 154.—Mouse liver. Ito cell tumor. Higher magnification of Figure 153. FIGURE 155.—Mouse liver. Histiocytic sarcoma. Perivascular infiltration. FIGURE 156.—Mouse liver. Hemangioma.

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FIGURE 157.—Mouse liver. Hemangioma. High magnification of Figure 156. FIGURE 158.—Mouse liver. Hemangiosarcoma. FIGURE 159.—Mouse liver. Hemangiosarcoma. Higher magnification of Figure 158. FIGURE 160.—Mouse liver. Hemangiosarcoma. Higher magnification of Figure 159. FIGURE 161.—Mouse liver. Extramedullary hematopoiesis. FIGURE 162.—Mouse liver. Extramedullary hematopoiesis. Myelopoiesis.

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FIGURE 163.—Mouse liver. Intrahepatic erythrocytes. FIGURE 164.—Rat liver. Pancreatic acinar metaplasia. FIGURE 165.—Rat liver. Ectopic acinar pancreas. FIGURE 166.—Rat liver. Hepatocyte glandular metaplasia. FIGURE 167.—Rat liver. Hepatocyte glandular metaplasia. FIGURE 168.—Mouse liver. Subintimal intravascular hepatocyte proliferation.

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FIGURE 169.—Mouse gallbladder. Heterotopic acinar pancreas. FIGURE 170.—Mouse gallbladder. Heterotopic acinar pancreas. Higher magnification of Figure 169. FIGURE 171.—Mouse gallbladder. Hyalinosis. Part of a biliary calculus is present. FIGURE 172.—Mouse gallbladder. Hyalinosis with crystal formation. FIGURE 173.—Mouse gallbladder. Hyalinosis and mucosal hyperplasia. Higher magnification of Figure 172. FIGURE 174.—Mouse gallbladder. Hyalinosis with crystal formation. Higher magnification of Figure 172.

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FIGURE 175.—Mouse gallbladder. Biliary calculus (biliary stone). FIGURE 176.—Mouse gallbladder. Hyalinosis and biliary calculus. FIGURE 177. Mouse gallbladder. Cholecystitis. FIGURE 178.—Mouse gallbladder. Submucosal edema. FIGURE 179.—Mouse gallbladder. Mucosal hyperplasia. FIGURE 180.—Mouse gallbladder. Mucosal hyperplasia. Higher magnification of Figure 179.

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FIGURE 181.—Mouse gallbladder. Papillary adenoma. FIGURE 182.—Mouse gallbladder. Papillary adenoma. FIGURE 183.—Mouse gallbladder. Papillary adenoma. Higher magnification of Figure 182.

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